Nucleic acids encoding oryza sativa nuclear cap binding protein 80 and methods of use

ABSTRACT

An isolated polynucleotide encoding a full-length rice cap binding protein 80 (CBP80) is provided. The reduction in expression or activity of CBP80 in a transgenic rice plant cell may be accomplished by antisense sequences which can inhibit expression or activity of endogenous CBP80 in a rice plant cell which may lead to alterations in hormone signaling.

This is a national stage application under 35 U.S.C. § 371 ofInternational Application No. PCT/EP02/03809, filed Apr. 5, 2002, whichis entitled to the benefit of United States Patent Application No.60/282,370, filed Apr. 6, 2001, both of which are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

The present invention generally relates to the field of plant molecularbiology, and more specifically to monocot nuclear cap binding protein 80(CBP80) genes.

BACKGROUND OF THE INVENTION

Unpredictable rainfall, increases in soil salinity, and low temperatureat the beginning or end of the growing season often result in decreasedplant growth and crop productivity. These three environmental factorsshare at least one element of stress, and that is water deficit ordehydration.

Drought is a significant problem in agriculture today. Over the last 40years, for example, drought accounted for 74% of the total U.S. croplosses of corn (U.S. Department of Agriculture, 1990. AgriculturalStatistics. U.S. Government Printing Office, Washington, D.C.). Tosustain productivity under adverse environmental conditions, it isimportant to provide crops with a genetic basis for coping with waterdeficit, for example, by breeding water retention and/or droughttolerance mechanisms into crops so that they can grow and yield underthese adverse conditions.

When the rate of transpiration exceeds that of water uptake or supply,water deficit occurs and wilting symptoms appear. The responses ofplants to water deficits include leaf rolling and shedding, stomataclosure, leaf temperature increases, and wilting. Metabolism is alsoprofoundly affected. General protein synthesis is inhibited andsignificant increases in certain amino acid pools, such as proline,become apparent (Barnett et al., 1966). During these water deficitperiods, the photosynthetic rate decreases with the ultimate result ofloss in yield (Boyer, 1976). If carried to an extreme, severe waterdeficits result in death of the plant.

Moreover, fresh water is increasingly becoming a scarce and threatenedresource in large part due to agricultural production (Serageldin,1995). Some studies have suggested that partial reduction in stomatalapertures could optimize CO₂ and H₂O exchange, particularly in light ofrising atmospheric CO₂ levels (Morison et al., 1987) and thus optimizeCO₂ flow into leaves for photosynthesis and water loss throughtranspiration. Classical studies showed that light-induced stomatalopening is mediated by K⁺ and anion accumulation in guard cells

A network of ion channels in the plasma membrane and the vacuolarmembrane of guard cells, which controls stomatal movements, has beencharacterized. These ion channels are targets of early signalingbranches and provide probes to identify and characterize upstreamtransducers. Guard cell signaling integrates water status, hormonalstimuli, light, CO₂ levels and other environmental conditions toregulate stomatal apertures. Thus, guard cells are a well-developedmodel system for understanding how components interact within asignaling network in a single cell as they respond at the single celllevel to physiological stimulation, allowing cell biological analyses inresponse to diverse stimuli. Guard cells respond to most of theclassical plant hormones, which illustrates that unidentified receptorsand early signaling mechanisms function in these cells.

Abscisic acid (ABA) mediates stress tolerance responses in higherplants, and is a key signal compound that regulates stomatal poreclosure in leaves, a process requiring ion channel modulation bycytoplasmic proteins (Schroeder et al., 2001). Several soluble signalingproteins have been proposed to regulate guard cell ABA signaltransduction (Schroeder et al., 2001). For example, the Arabidopsisfarnesyltransferase (FTase) β subunit, encoded by ERA1, has been shownto be a negative regulator of ABA signal transduction in seeds and guardcells (Pei et al., 1998). Deletion of the ERA1 gene in Arabidopsiscauses ABA hypersensitivity of both anion channel activation andstomatal closing, and during drought treatment era1 plants show reducedwilting and enhanced stomatal closure. Notably, when era1 plants arewatered, the stomatal pores open, although to a lesser extent than inwild type, allowing CO₂ influx and growth. Intragenic suppressor mutantsof abil-1 (abil-1R) also show reduced water loss during drought;however, era1 and abil-1R alleles have additional growth anddevelopmental phenotypes, e.g., ERA1 null mutant plants alsodemonstrated slowed growth (Pei et al. 1998).

Thus, downregulation of negative regulators or upregulation of positiveregulators in guard cells using guard cell-specific andstress-responsive promoters, or isolation of guard cell-specificmutants, may allow engineering of plants that lose less water duringdrought. Some mutations may constitutively reduce stomatal opening,which could reduce the water requirements of horticultural plants andturf grass, or in agricultural regions of marginal fresh wateravailability, or during drought so as to slow desiccation and damage.For plants growing in humid regions, weakening of ABA signaltransduction components may enhance stomatal opening and CO₂ intake forcarbon fixation and growth (Schroeder et al., 2001). However,conventional high-yield breeding approaches may have contributed toselection of crop plants with reduced stomatal ABA responsiveness,because genes controlling guard cell signaling are also expressed inother tissues and control other yield parameters.

Thus, what is needed is the further identification of plant genes, e.g.,monocot genes, that are negative regulators of ABA signal transduction,which are useful to engineer drought hardiness into plants.

SUMMARY OF THE INVENTION

The invention relates to plants having altered, e.g., reduced,expression and/or activity of endogenous cap binding protein 80 (CBP80),including monocot CBP80, and methods of preparing and using thoseplants. The reduction in expression and/or activity of CBP80 in a cellmay be accomplished by expression of antisense sequences, a ribozyme,RNAi, a dominant negative CBP80, or by a CBP80 gene knock out, e.g., viatargeted gene disruption, and the like, which can specifically inhibitexpression and/or activity of endogenous CBP80 in a host cell. Thus, thepresent invention provides an isolated nucleic acid molecule(polynucleotide) having a nucleotide sequence that encodes a full-lengthCBP80 or a fragment (portion) thereof which preferably encodes apartial-length CBP80 with substantially the same activity, e.g., atleast 50%, preferably 70% and more preferably 90 to 95%, the activity,of the full-length CBP80. The nucleotide sequence preferably is obtainedor isolated from a plant, for instance a monocot, DNA that encodes apolypeptide which is substantially similar, e.g., has at least 68%, 69%,and preferably has at least 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, andeven 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, up to atleast 99%, amino acid sequence identity, to SEQ ID NO:2 or a fragment(portion) thereof which is a partial-length polypeptide havingsubstantially the same activity as SEQ ID NO:2, and optionally includesthe sequence PPTISQS (SEQ ID NO:20). In one embodiment, the nucleotidesequence encodes a CBP80 polypeptide of less than 900 residues in lengthand/or encodes a CBP80 polypeptide having at least 96% identity to SEQID NO:2.

The present invention also provides an isolated nucleic acid moleculehaving a nucleotide sequence, preferably obtained or isolated frommonocot DNA. Preferably, the nucleotide sequence is substantiallysimilar, i.e., has at least 65%, 66%, 67%, 68%, 69%, and preferably hasat least 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even 90% or more, e.g.,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, up to at least 99%, nucleic acidsequence identity, to SEQ ID NO:1, the complement thereof, a fragment(portion) thereof which encodes a partial-length polypeptide havingsubstantially the same activity as SEQ ID NO:2, or a fragment useful toprepare a construct or vector to decrease or eliminate expression of thecorresponding endogenous gene. Preferably, the term “substantiallysimilar” when used herein with respect to a nucleotide sequence meansthat the nucleotide sequence is part of a gene which encodes apolypeptide having substantially the same structure and function as thepolypeptide encoded by the reference nucleotide sequence. The term“substantially similar” thus includes sequences which have beenmodified, e.g., to optimize expression in particular cells, as well asnucleotide sequences encoding a variant polypeptide comprising one ormore amino acid to substitutions relative to the (unmodified)polypeptide encoded by the reference sequence, which substitution(s)does not alter the activity of the variant polypeptide relative to theunmodified polypeptide.

Sequence comparisons may be carried out using a Smith-Waterman sequencealignment algorithm (see e.g. Waterman (1995) or on the World Wide Webat hto.usc.edu/software/segaln/index.html). The localS program, version1.16, is preferably used with following parameters: match: 1, mismatchpenalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2. Further, anucleotide sequence that is “substantially similar” to a referencenucleotide sequence hybridizes to the reference nucleotide sequence in7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. withwashing in 2×SSC, 0.1% SDS at 50° C., more desirably in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in1×SSC, 0.1% SDS at 50° C., more desirably still in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.5×SSC,0.1% SDS at 50° C., preferably in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C.,more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mMEDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C.

Hence, the isolated polynucleotides of the invention include theorthologs of the Oryza sativa sequences disclosed herein, i.e.,polynucleotides in organisms other than Oryza sativa, including, but notlimited to, monocots other than Oryza, preferably cereal plants, e.g.,corn or wheat. An ortholog is a gene from a different species thatencodes a product having the same function, e.g., catalyzes the samereaction, as the product encoded by a gene from a reference organism.Databases such GenBank may be employed to identify sequences related tothe Oryza sequences, e.g., orthologs in cereal crops such as corn.Alternatively, recombinant DNA techniques such as hybridization or PCRmay be employed to identify sequences related to the Oryza sequences.The encoded ortholog products likely have at least 70% sequence identityto each other. Hence, the invention includes an isolated polynucleotidecomprising a nucleotide sequence encoding a polypeptide having at least70% identity to a polypeptide encoded by the Oryza CBP80 sequencedisclosed herein. However, an ortholog includes polypeptides having lessthan 65% amino acid sequence identity but which has the same or similarfunction as the reference polypeptide. The invention also providesantisense polynucleotides corresponding to the genes, e.g., open readingframes or fragments thereof, identified herein. Also provided areexpression cassettes, e.g., recombinant vectors, and host cells,comprising the polynucleotide of the invention in either sense orantisense orientation. Thus, the present invention further provides aconstruct, vector or expression cassette comprising the polynucleotideof the invention, either in sense or antisense, or both, optionallylinked to a promoter. The vector may be a plasmid. Such constructs,vectors or expression cassettes, when present in a plant, plant cell orplant tissue result in transcription of the linked polynucleotide in theplant and/or result in a gene knock out, so as to reduce or inhibit, andpreferably eliminate, expression, of the endogenous CBP80 gene.

The constructs, expression cassettes or vectors of the invention mayoptionally include other regulatory sequences, e.g., transcriptionterminator sequences, introns and/or enhancers, and may be contained ina host cell. Preferred regulatory sequences include those which areexpressed in leaf and more preferably in guard cells of a plant.Promoters for expression in guard cells include but are not limited tothe KAT1 promoter (Nakamura et al., 1995), the ERA1 promoter and the ADPglucose phosphorylase promoter (Muller-Rober et al., 1994). Theconstruct, expression cassette or vector may augment the genome of atransformed plant or may be maintained extrachromosomally. Theconstruct, expression cassette or vector may further have a Ti plasmidand be contained in an Agrobacterium tumefaciens cell; it may be carriedon a microparticle, wherein the microparticle is suitable for ballistictransformation of a plant cell; or it may be contained in a plant cellprotoplast. Further, the construct, expression cassette or vector can becontained in a plant, plant cell or plant tissue from a dicot or amonocot. In particular, the plant may be a cereal plant.

The present invention further provides a method of augmenting a plantgenome by contacting plant cells with a polynucleotide of the invention,e.g., one isolatable or obtained from a plant gene encoding apolypeptide that is substantially similar to SEQ ID NO:2, so as to yieldtransformed plant cells; and regenerating the transformed plant cells toprovide a differentiated transformed plant, wherein the differentiatedtransformed plant xpresses the nucleic acid molecule in the cells of theplant. The nucleic acid molecule may be present in the nucleus,chloroplast, mitochondria and/or plastid of the cells of the plant. Thepresent invention also provides a transgenic plant prepared by thismethod, a seed from such a plant and progeny plants from such a plantincluding hybrids and inbreds. Preferred transgenic plants aretransgenic maize, soybean, barley, alfalfa, sunflower, canola, soybean,cotton, peanut, sorghum, tobacco, sugarbeet, rice, wheat, rye,turfgrass, millet, sugarcane, tomato, or potato.

The invention also provides a method of plant breeding, e.g., to preparea crossed fertile transgenic plant. The method comprises crossing afertile transgenic plant comprising a particular nucleic acid moleculeof the invention with itself or with a second plant, e.g., one lackingthe particular nucleic acid molecule, to prepare the seed of a crossedfertile transgenic plant comprising the particular nucleic acidmolecule. The seed is then planted to obtain a crossed fertiletransgenic plant. The plant may be a monocot or a dicot. In a particularembodiment, the plant is a cereal plant.

The crossed fertile transgenic plant may have the particular nucleicacid molecule inherited through a female parent or through a maleparent. The second plant may be an inbred plant. The crossed fertiletransgenic may be a hybrid. Also included within the present inventionare seeds of any of these crossed fertile transgenic plants.

The various breeding steps are characterized by well-defined humanintervention such as selecting the lines to be crossed, directingpollination of the parental lines, or selecting appropriate progenyplants. Depending on the desired properties different breeding measuresare taken. The relevant techniques are well known in the art and includebut are not limited to hybridization, inbreeding, backcross breeding,multiline breeding, variety blend, interspecific hybridization,aneuploid techniques, etc. Hybridization techniques also include thesterilization of plants to yield male or female sterile plants bymechanical, chemical or biochemical means. Cross pollination of a malesterile plant with pollen of a different line assures that the genome ofthe male sterile but female fertile plant will uniformly obtainproperties of both parental lines. Thus, the transgenic plants accordingto the invention can be used for the breeding of improved plant linesthat for example increase the effectiveness of conventional methods suchas herbicide or pesticide treatment or allow to dispense with saidmethods due to their modified genetic properties. Alternatively newcrops with improved stress tolerance can be obtained that, due to theiroptimized genetic “equipment”, yield harvested product of better qualitythan products that were not able to tolerate comparable adversedevelopmental conditions.

The polynucleotides of the invention, their encoded polypeptides andcompositions thereof, are useful to alter abscisic acid signaling,stomatal pore opening or closing (frequency or size of opening), waterloss, senescence, anion channel activity, drought tolerance, or anycombination thereof, in a plant by over- or under- or knock out, ofexpression of CBP80. Methods to detect abscisic acid signaling, stomatalpore opening or closing, water loss, senescence, anion channel activity,or drought tolerance in a plant or plant cell are well known to the art,see, e.g., WO 01/96585. The compositions of the invention include full-or partial-length plant polynucleotides and the amino acid sequences forthe full- or partial-length polypeptides encoded thereby. Methods of theinvention involve stably transforming a plant with one or more of atleast a portion of these polynucleotides comprising an open readingframe operably linked to a promoter capable of driving expression, e.g.,antisense, of that polynucleotide in a plant cell. In anotherembodiment, the methods of the invention involve stably transforming aplant with a polynucleotide of the invention to yield a plant, theindogenous CBP80 gene(s) of which is disrupted, i.e., a CBP80 “knockout” plant. By “portion” or “fragment”, as it relates to a nucleotidesequence of the invention, when it is linked to other sequences forexpression, is meant a sequence having at least 80 nucleotides, morepreferably at least 150 nucleotides, and still more preferably at least700 nucleotides. If not employed for expressing, a “portion” or“fragment” means at least 9, preferably 12, more preferably 15, evenmore preferably at least 20, consecutive nucleotides, e.g., probes andprimers (oligonucleotides), corresponding to the nucleotide sequence.

The method also comprises introducing to a plant, plant cell, or planttissue an expression cassette comprising an inhibitor of CBP80, e.g.,antisense sequences, a ribozyme, RNAi, or another small molecule whichspecifically inhibits CBP80 or its expression, so as to yield atransformed differentiated plant, transformed cell or transformedtissue. Transformed cells or tissue can be regenerated to provide atransformed differentiated plant. The transformed differentiated plantpreferably expresses the inhibitor in an amount that enhances ABAsignaling in the plant, enhances stomatal closing or frequency,increases drought tolerance, increases anion channel activity, or anycombination thereof. The present invention also provides a transformedplant prepared by the method, progeny and seed thereof. Further, thepolynucleotide of the invention, when present in a plant in a mannerwhich inhibits, reduces or eliminates expression of endogenous CBP80 soas to result in a drought tolerant plant, may be employed as aselectable marker.

A method to shuffle the polynucleotide of the invention is provided.This method involves fragmentation of a nucleic acid corresponding to apolynucleotide listed in SEQ ID NO:1, the orthologs thereof, and thecorresponding genes, followed by religation. This method allows for theproduction of polypeptides having altered activity relative to thenative form of the polypeptide. Accordingly, the invention providescells and transgenic plants containing nucleic acid segments producedthrough shuffling that encode polypeptides having altered activityrelative to the corresponding native polypeptide.

A computer readable medium, e.g., a magnetic tape, optical disk, CD-ROM,random access memory, volatile memory, non-volatile memory, or bubblememory, containing the nucleic acid sequences of the invention as wellas methods of use for the computer readable medium are provided. Forexample, a computer readable medium can contain a nucleic acid moleculethat has at least 70% nucleic acid sequence identity to SEQ ID NO:1 orthe complement thereof. This medium allows a nucleic acid segmentcorresponding to a nucleic acid sequence listed in SEQ ID NO:1 to beused as a reference sequence to search against databases. This mediumalso allows for computer-based manipulation of a nucleic acid sequencecorresponding to a nucleic acid sequence listed in SEQ ID NOs:1, and thecorresponding gene and polypeptide encoded by the nucleic acid sequence.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The term “gene” is used broadly to refer to any segment of nucleic acidassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.For example, gene refers to a nucleic acid fragment that expresses mRNAor functional RNA, or encodes a specific protein, and which includesregulatory sequences. Genes also include nonexpressed DNA segments that,for example, form recognition sequences for other proteins. Genes can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters.

The term “native” or “wild type” gene refers to a gene that is presentin the genome of an untransformed cell, i.e., a cell not having a knownmutation.

A “marker gene” encodes a selectable or screenable trait.

The term “chimeric gene” refers to any gene that contains 1) DNAsequences, including regulatory and coding sequences, that are not foundtogether in nature, or 2) sequences encoding parts of proteins notnaturally adjoined, or 3) parts of promoters that are not naturallyadjoined. Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orcomprise regulatory sequences and coding sequences derived from the samesource, but arranged in a manner different from that found in nature.

A “transgene” refers to a gene that has been introduced into the genomeby transformation and is stably maintained. Transgenes may include, forexample, genes that are either heterologous or homologous to the genesof a particular plant to be transformed. Additionally, transgenes maycomprise native genes inserted into a non-native organism, or chimericgenes. The term “endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism but that is introduced by genetransfer.

An “oligonucleotide” corresponding to a nucleotide sequence of theinvention, e.g., for use in probing or amplification reactions, may beabout 30 or fewer nucleotides in length (e.g., 9, 12, 15, 18, 20, 21 or24, or any number between 9 and 30). Generally specific primers areupwards of 14 nucleotides in length. For optimum specificity and costeffectiveness, primers of 16 to 24 nucleotides in length may bepreferred. Those skilled in the art are well versed in the design ofprimers for use processes such as PCR. If required, probing can be donewith entire restriction fragments of the gene disclosed herein which maybe 100's or even 1000's of nucleotides in length.

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein.

The nucleotide sequences of the invention can be introduced into anyplant. The genes to be introduced can be conveniently used in expressioncassettes for introduction and expression in any plant of interest. Suchexpression cassettes will comprise the transcriptional initiation regionof the invention linked to a nucleotide sequence of interest. Preferredpromoters include constitutive, tissue-specific, developmental-specific,inducible and/or viral promoters. Such an expression cassette isprovided with a plurality of restriction sites for insertion of the geneof interest to be under the transcriptional regulation of the regulatoryregions. The expression cassette may additionally contain selectablemarker genes. The cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational termination regionfunctional in plants. The termination region may be native with thetranscriptional initiation region, may be native with the DNA sequenceof interest, or may be derived from another source. Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also, Guerineau et al., 1991; Proudfoot, 1991; Sanfacon et al.,1991; Mogen et al., 1990; Munroe et al., 1990; Ballas et al., 1989;Joshi et al., 1987.

“Coding sequence” refers to a DNA or RNA sequence that codes for aspecific amino acid sequence and excludes the non-coding sequences. Itmay constitute an “uninterrupted coding sequence”, i.e., lacking anintron, such as in a cDNA or it may include one or more introns boundedby appropriate splice junctions. An “intron” is a sequence of RNA whichis contained in the primary transcript but which is removed throughcleavage and re-ligation of the RNA within the cell to create the maturemRNA that can be translated into a protein.

The terms “open reading frame” and “ORF” refer to the amino acidsequence encoded between translation initiation and termination codonsof a coding sequence. The terms “initiation codon” and “terminationcodon” refer to a unit of three adjacent nucleotides (‘codon’) in acoding sequence that specifies initiation and chain termination,respectively, of protein synthesis (mRNA translation).

A “functional RNA” refers to an antisense RNA, ribozyme, or other RNAthat is not translated. The term “RNA transcript” refers to the productresulting from RNA polymerase catalyzed transcription of a DNA sequence.When the RNA transcript is a perfect complementary copy of the DNAsequence, it is referred to as the primary transcript or it may be a RNAsequence derived from posttranscriptional processing of the primarytranscript and is referred to as the mature RNA. “Messenger RNA” (mRNA)refers to the RNA that is without introns and that can be translatedinto protein by the cell. “cDNA” refers to a single- or adouble-stranded DNA that is complementary to and derived from mRNA.

“Regulatory sequences” and “suitable regulatory sequences” each refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences includeenhancers, promoters, translation-leader sequences, introns, andpolyadenylation signal sequences. They include natural and syntheticsequences as well as sequences which may be a combination of syntheticand natural sequences. As is noted above, the term “suitable regulatorysequences” is n t limited to promoters.

“5′non-coding sequence” refers to a nucleotide sequence located 5′(upstream) to the coding sequence. It is present in the fully processedmRNA upstream of the initiation codon and may affect processing of theprimary transcript to mRNA, mRNA stability or translation efficiency(Turner et al., 1995). “3′ non-coding sequence” refers to nucleotidesequences located 3′ (downstream) to a coding sequence and includepolyadenylation signal sequences and other sequences encoding regulatorysignals capable of affecting mRNA processing or gene expression. Thepolyadenylation signal is usually characterized by affecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNAprecursor. The use of different 3′ non-coding sequences is exemplifiedby Ingelbrecht et al., 1989.

The term “translation leader sequence” refers to that DNA sequenceportion of a gene between the promoter and coding sequence that istranscribed into RNA and is present in the fully processed mRNA upstream(5) of the translation start codon. The translation leader sequence mayaffect processing of the primary transcript to mRNA, mRNA stability ortranslation efficiency.

The term “mature” protein refers to a post-translationally processedpolypeptide without its signal peptide. “Precursor” protein refers tothe primary product of translation of an mRNA. “Signal peptide” refersto the amino terminal extension of a polypeptide, which is translated inconjunction with the polypeptide forming a precursor peptide and whichis required for its entrance into the secretory pathway. The term“signal sequence” refers to a nucleotide sequence that encodes thesignal peptide.

The term “intracellular localization sequence” refers to a nucleotidesequence that encodes an intracellular targeting signal. An“intracellular targeting signal” is an amino acid sequence that istranslated in conjunction with a protein and directs it to a particularsubs cellular compartment. “Endoplasmic reticulum (ER) stop transitsignal” refers to a carboxy-terminal extension of a polypeptide, whichis translated in conjunction with the polypeptide and causes a proteinthat enters the secretory pathway to be retained in the ER. “ER stoptransit sequence” refers to a nucleotide sequence that encodes the ERtargeting signal. Other intracellular targeting sequences encodetargeting signals active in seeds and/or leaves and

“Promoter” refers to a nucleotide sequence, usually upstream (5) to itscoding sequence, which controls the expression of the coding sequence byproviding the recognition for RNA polymerase and other factors requiredfor proper transcription. “Promoter” includes a minimal promoter that isa short DNA sequence comprised of a TATA box and other sequences thatserve to specify the site of transcription initiation, to whichregulatory elements are added for control of expression. “Promoter” alsorefers to a nucleotide sequence that includes a minimal promoter plusregulatory elements that is capable of controlling the expression of acoding sequence or functional RNA. This type of promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue specificity of a promoter. It is capable ofoperating in both orientations (normal or flipped), and is capable offunctioning even when moved either upstream or downstream from thepromoter. Both enhancers and other upstream promoter elements bindsequence-specific DNA-binding proteins that mediate their effects.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even be comprised of synthetic DNA segments. A promoter mayalso contain DNA sequences that are involved in the binding of proteinfactors which control the effectiveness of transcription initiation inresponse to physiological or developmental conditions.

The “initiation site” is the position surrounding the first nucleotidethat is part of the transcribed sequence, which is also defined asposition +1. With respect to this site all other sequences of the geneand its controlling regions are numbered. Downstream sequences (i.e.,further protein encoding sequences in the 3′ direction) are denominatedpositive, while upstream sequences (mostly of the controlling regions inthe 5′ direction) are denominated negative.

Promoter elements, particularly a TATA element, that are inactive orthat have greatly reduced promoter activity in the absence of upstreamactivation are referred to as “minimal or core promoters.” In thepresence of a suitable transcription factor, the minimal promoterfunctions to permit transcription. A “minimal or core promoter” thusconsists only of all basal elements needed for transcription initiation,e.g., a TATA box and/or an initiator.

“Constitutive expression” refers to expression using a constitutive orregulated promoter. “Conditional” and “regulated expression” refer toexpression controlled by a regulated promoter.

“Constitutive promoter” refers to a promoter that is able to express theopen reading frame (ORF) that it controls in all or nearly all of theplant tissues during all or nearly all developmental stages of theplant. Each of the transcription-activating elements do not exhibit anabsolute tissue-specificity, but mediate transcriptional activation inmost plant parts at a level of ≧1% of the level reached in the part ofthe plant in which transcription is most active.

“Regulated promoter” refers to promoters that direct gene expression notconstitutively, but in a temporally- and/or spatially-regulated manner,and includes both tissue-specific and inducible promoters. It includesnatural and synthetic sequences as well as sequences which may be acombination of synthetic and natural sequences. Different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. New promoters of various types useful in plantcells are constantly being discovered, numerous examples may be found inthe compilation by Okamuro et al. (1989). Typical regulated promotersuseful in plants include but are not limited to safener-induciblepromoters, promoters derived from the tetracycline-inducible system,promoters derived from salicylate-inducible systems, promoters derivedfrom alcohol-inducible systems, promoters derived fromglucocorticoid-inducible system, promoters derived frompathogen-inducible systems, and promoters derived fromecdysone-inducible systems.

“Tissue-specific promoter” refers to regulated promoters that are notexpressed in all plant cells but only in one or more cell types inspecific organs (such as leaves or seeds), specific tissues (such asembryo or cotyledon), or specific cell types (such as leaf parenchyma orseed storage cells). These also include promoters that are temporallyregulated, such as in early or late embryogenesis, during fruit ripeningin developing seeds or fruit, in fully differentiated leaf, or at theonset of senescence.

“Inducible promoter” refers to those regulated promoters that can beturned on in one or more cell types by an external stimulus, such as achemical, light, hormone, stress, or a pathogen.

“Operably-linked” refers to the association of nucleic acid sequences onsingle nucleic acid fragment so that the function of one is affected bythe other. For example, a regulatory DNA sequence is said to be“operably linked to” or “associated with” a DNA sequence that codes foran RNA or a polypeptide if the two sequences are situated such that theregulatory DNA sequence affects expression of the coding DNA sequence(i.e., that the coding sequence or functional RNA is under thetranscriptional control of the promoter). Coding sequences can beoperably-linked to regulatory sequences in sense or antisenseorientation.

“Expression” refers to the transcription and/or translation of anendogenous gene, ORF or portion thereof, or a transgene in plants. Forexample, in the case of antisense constructs, expression may refer tothe transcription of the antisense DNA only. In addition, expressionrefers to the transcription and stable accumulation of sense (mRNA) orfunctional RNA. Expression may also refer to the production of protein.

“Specific expression” is the expression of gene products which islimited to one or a few plant tissues (spatial limitation) and/or to oneor a few plant developmental stages (temporal limitation). It isacknowledged that hardly a true specificity exists: promoters seem to bepreferably switch on in some tissues, while in other tissues there canbe no or only little activity. This phenomenon is known as leakyexpression. However, with specific expression in this invention is meantpreferable expression in one or a few plant tissues.

The “expression pattern” of a promoter (with or without enhancer) is thepattern of expression levels which shows where in the plant and in whatdevelopmental stage transcription is initiated by said promoter.Expression patterns of a set of promoters are said to be complementarywhen the expression pattern of one promoter shows little overlap withthe expression pattern of the other promoter. The level of expression ofa promoter can be determined by measuring the ‘steady state’concentration of a standard transcribed reporter mRNA. This measurementis indirect since the concentration of the reporter mRNA is dependentnot only on its synthesis rate, but also on the rate with which the mRNAis degraded. Therefore, the steady state level is the product ofsynthesis rates and degradation rates.

The rate of degradation can however be considered to proceed at a fixedrate when the transcribed sequences are identical, and thus this valuecan serve as a measure of synthesis rates. When promoters are comparedin this way techniques available to those skilled in the art arehybridization S1-RNAse analysis, northern blots and competitive RT-PCR.This list of techniques in no way represents all available techniques,but rather describes commonly used procedures used to analyzetranscription activity and expression levels of mRNA.

The analysis of transcription start points in practically all promotershas revealed that there is usually no single base at which transcriptionstarts, but rather a more or less clustered set of initiation sites,each of which accounts for some start points of the mRNA. Since thisdistribution varies from promoter to promoter the sequences of thereporter mRNA in each of the populations would differ from each other.Since each mRNA species is more or less prone to degradation, no singledegradation rate can be expected for different reporter mRNAs. It hasbeen shown for various eukaryotic promoter sequences that the sequencesurrounding the initiation site (‘initiator’) plays an important role indetermining the level of RNA expression directed by that specificpromoter. This includes also part of the transcribed sequences. Thedirect fusion of promoter to reporter sequences would therefore lead tosuboptimal levels of transcription.

A commonly used procedure to analyze expression patterns and levels isthrough determination of the ‘steady state’ level of proteinaccumulation in a cell. Commonly used candidates for the reporter gene,known to those skilled in the art are ∃-glucuronidase (GUS),chloramphenicol acetyl transferase (CAT) and proteins with fluorescentproperties, such as is green fluorescent protein (GFP) from Aequoravictoria. In principle, however, many more proteins are suitable forthis purpose, provided the protein does not interfere with essentialplant functions. For quantification and determination of localization anumber of tools are suited. Detection systems can readily be created orare available which are based on, e.g., immunochemical, enzymatic,fluorescent detection and quantification. Protein levels can bedetermined in plant tissue extracts or in intact tissue using in situanalysis of protein expression.

Generally, individual transformed lines with one chimeric promoterreporter construct will vary in their levels of expression of thereporter gene. Also frequently observed is the phenomenon that suchtransformants do not express any detectable product (RNA or protein).The variability in expression is commonly ascribed to ‘positioneffects’, although the molecular mechanisms underlying this inactivityare usually not clear.

The term “average expression” is used here as the average level ofexpression found in all lines that do express detectable amounts ofreporter gene, so leaving out of the analysis plants that do not expressany detectable reporter mRNA or protein.

“Non-specific expression” refers to constitutive expression or lowlevel, basal (‘leaky’) expression in nondesired cells or tissues from a‘regulated promoter’.

“Altered levels” refers to the level of expression in transgenicorganisms that differs from that of normal or untransformed organisms.

“Overexpression” refers to the level of expression in transgenic cellsor organisms that exceeds levels of expression in normal oruntransformed (nontransgenic) cells or organisms.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of protein from anendogenous gene or a transgene.

“Co-suppression” and “transwitch” each refer to the production of senseRNA transcripts capable of suppressing the expression of identical orsubstantially similar transgene or endogenous genes (U.S. Pat. No.5,231,020).

“Gene silencing” refers to homology-dependent suppression of viralgenes, transgenes, or endogenous nuclear genes. Gene silencing may betranscriptional, when the suppression is due to decreased transcriptionof the affected genes, or post-transcriptional, when the suppression isdue to increased turnover (degradation) of RNA species homologous to theaffected genes (English et al., 1996). Gene silencing includesvirus-induced gene silencing (Ruiz et al. 1998).

“Silencing suppressor” gene refers to a gene whose expression leads tocounteracting gene silencing and enhanced expression of silenced genes.Silencing suppressor genes may be of plant, non-plant, or viral origin.Examples include, but are not limited to HC-Pro, P1-HC-Pro, and 2bproteins. Other examples include one or more genes in TGMV-B genome.

The terms “heterologous DNA sequence,” “exogenous DNA segment” or“heterologous nucleic acid,” as used herein, each refer to a sequencethat originates from a source foreign to the particular host cell or, iffrom the same source, is modified from its original form. Thus, aheterologous gene in a host cell includes a gene that is endogenous tothe particular host cell but has been modified through, for example, theuse of DNA shuffling. The terms also include non-naturally occurringmultiple copies of a naturally occurring DNA sequence. Thus, the termsrefer to a DNA segment that is foreign or heterologous to the cell, orhomologous to the cell but in a position within the host cell nucleicacid in which the element is not ordinarily found. Exogenous DNAsegments are expressed to yield exogenous polypeptides. A “homologous”DNA sequence is a DNA sequence that is naturally associated with a hostcell into which it is introduced.

“Homologous to” in the context of nucleotide sequence Identity refers tothe similarity between the nucleotide sequence of two nucleic acidmolecules or between the amino acid sequences of two protein molecules.Estimates of such homology are provided by either DNA-DNA or DNA-RNAhybridization under conditions of stringency as is well understood bythose skilled in the art (as described in Haines and Higgins (eds.),Nucleic Acid Hybridization, IRL Press, Oxford, U.K.), or by thecomparison of sequence similarity between two nucleic acids or proteins.

The term “substantially similar” refers to nucleotide and amino acidsequences that represent functional and/or structural equivalents ofArabidopsis sequences disclosed herein. For example, altered nucleotidesequences which simply reflect the degeneracy of the genetic code butnonetheless encode amino acid sequences that are identical to aparticular amino acid sequence are substantially similar to theparticular sequences. In addition, amino acid sequences that aresubstantially similar to a particular sequence are those wherein overallamino acid identity is at least 65% or greater to the instant sequences.Modifications that result in equivalent nucleotide or amino acidsequences are well within the routine skill in the art. Moreover, theskilled artisan recognizes that equivalent nucleotide sequencesencompassed by this invention can also be defined by their ability tohybridize, under low, moderate and/or stringent conditions (e.g.,0.1×SSC, 0.1% SDS, 65° C.), with the nucleotide sequences that arewithin the literal scope of the instant claims.

“Target gene” refers to a gene on the replicon that expresses thedesired target coding sequence, functional RNA, or protein. The targetgene is not essential for replicon replication. Additionally, targetgenes may comprise native non-viral genes inserted into a non-nativeorganism, or chimeric genes, and will be under the control of suitableregulatory sequences. Thus, the regulatory sequences in the target genemay come from any source, including the virus. Target genes may includecoding sequences that are either heterologous or homologous to the genesof a particular plant to be transformed. However, target genes do notinclude native viral genes. Typical target genes include, but are notlimited to genes encoding a structural protein, a seed storage protein,a protein that conveys herbicide resistance, and a protein that conveysinsect resistance. Proteins encoded by target genes are known as“foreign proteins”. The expression of a target gene in a plant willtypically produce an altered plant trait.

The term “altered plant trait” means any phenotypic or genotypic changein a transgenic plant relative to the wild-type or non-transgenic planthost. “Transcription Stop Fragment” refers to nucleotide sequences thatcontain one or more regulatory signals, such as polyadenylation signalsequences, capable of terminating transcription. Examples include the 3′non-regulatory regions of genes encoding nopaline synthase and the smallsubunit of ribulose bisphosphate carboxylase.

“Replication gene” refers to a gene encoding a viral replicationprotein. In addition to the ORF of the replication protein, thereplication gene may also contain other overlapping or non-overlappingORF(s), as are found in viral sequences in nature. While not essentialfor replication, these additional ORFs may enhance replication and/orviral DNA accumulation. Examples of such additional ORFs are AC3 and AL3in ACMV and TGMV geminiviruses, respectively.

“Chimeric trans-acting replication gene” refers either to a replicationgene in which the coding sequence of a replication protein is under thecontrol of a regulated plant promoter other than that in the nativeviral replication gene, or a modified native viral replication gene, forexample, in which a site specific sequence(s) is inserted in the 5′transcribed but untranslated region. Such chimeric genes also includeinsertion of the known sites of replication protein binding between thepromoter and the transcription start site that attenuate transcriptionof viral replication protein gene.

“Chromosomally-integrated” refers to the integration of a foreign geneor DNA construct into the host DNA by covalent bonds. Where genes arenot “chromosomally integrated” they may be “transiently expressed.”Transient expression of a gene refers to the expression of a gene thatis not integrated into the host chromosome but functions independently,either as part of an autonomously replicating plasmid or expressioncassette, for example, or as part of another biological system such as avirus.

“Production tissue” refers to mature, harvestable tissue consisting ofnon-dividing, terminally-differentiated cells. It excludes young,growing tissue consisting of germline, meristematic, andnot-fully-differentiated cells.

“Germline cells” refer to cells that are destined to be gametes andwhose genetic material is heritable.

“Trans-activation” refers to switching on of gene expression or repliconreplication by the expression of another (regulatory) gene in trans.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell, resulting in genetically stableinheritance. Host cells containing the transformed nucleic acidfragments are referred to as “transgenic” cells, and organismscomprising transgenic cells are referred to as “transgenic organisms”.Examples of methods of transformation of plants and plant cells includeAgrobacterium-mediated transformation (De Blaere et al., 1987) andparticle bombardment technology (Klein et al. 1987; U.S. Pat. No.4,945,050). Whole plants may be regenerated from transgenic cells bymethods well known to the skilled artisan (see, for example, Fromm etal., 1990).

“Transformed,” “transgenic,” and “recombinant” refer to a host organismsuch as a bacterium or a plant into which a heterologous nucleic acidmolecule has been introduced. The nucleic acid molecule can be stablyintegrated into the genome generally known in the art and are disclosedin Sambrook et al., 1989. See also Innis et al., 1995 and Gelfand, 1995;and Innis and Gelfand, 1999. Known methods of PCR include, but are notlimited to, methods using paired primers, nested primers, singlespecific primers, degenerate primers, gene-specific primers,vector-specific primers, partially mismatched primers, and the like. Forexample, “transformed,” “transformant,” and “transgenic” plants or callihave been through the transformation process and contain a foreign geneintegrated into their chromosome. The term “untransformed” refers tonormal plants that have not been through the transformation process.

“Transiently transformed” refers to cells in which transgenes andforeign DNA have been introduced (for example, by such methods asAgrobacterium-mediated transformation or biolistic bombardment), but notselected for stable maintenance.

“Stably transformed” refers to cells that have been selected andregenerated on a selection media following transformation.

“Transient expression” refers to expression in cells in which a virus ora transgene is introduced by viral infection or by such methods asAgrobacterium-mediated transformation, electroporation, or biolisticbombardment, but not selected for its stable maintenance.

“Genetically stable” and “heritable” refer to chromosomally-integratedgenetic elements that are stably maintained in the plant and stablyinherited by progeny through successive generations.

“Primary transformant” and “T0 generation” refer to transgenic plantsthat are of the same genetic generation as the tissue which wasinitially transformed (i.e., not having gone through meiosis andfertilization since transformation).

“Secondary transformants” and the “T1, T2, T3, etc. generations” referto transgenic plants derived from primary transformants through one ormore meiotic and fertilization cycles. They may be derived byself-fertilization of primary or secondary transformants or crosses ofprimary or secondary transformants with other transformed oruntransformed plants.

“Wild-type” refers to a virus or organism found in nature without anyknown mutation.

“Genome” refers to the complete genetic material of an organism.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base which is either a purine or pyrimidine.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides which have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., 1991; Ohtsuka et al., 1985;Rossolini et al. 1994). A “nucleic acid fragment” is a fraction of agiven nucleic acid molecule. In higher plants, deoxyribonucleic acid(DNA) is the genetic material while ribonucleic acid (RNA) is involvedin the transfer of information contained within DNA into proteins. Theterm “nucleotide sequence” refers to a polymer of DNA or RNA which canbe single- or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases capable of incorporation intoDNA or RNA polymers. The terms “nucleic acid” or “nucleic acid sequence”may also be used interchangeably with gene, cDNA, DNA and RNA encoded bya gene.

The invention encompasses isolated or substantially purified nucleicacid or protein compositions. In the context of the present invention,an “isolated” or “purified” DNA molecule or an “isolated” or “purified”polypeptide is a DNA molecule or polypeptide that, by the hand of man,exists apart from its native environment and is therefore not a productof nature. An isolated DNA molecule or polypeptide may exist in apurified form or may exist in a non-native environment such as, forexample, a transgenic host cell. For example, an “isolated” or“purified” nucleic acid molecule or protein, or biologically activeportion thereof, is substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized. Preferably, an “isolated” nucleic acid is free of sequences(preferably protein encoding sequences) that naturally flank the nucleicacid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleicacid molecule in genomic DNA of the cell from which the nucleic acid isderived. A protein that is substantially free of cellular materialincludes preparations of protein or polypeptide having less than about30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When theprotein of the invention, or biologically active portion thereof, isrecombinantly produced, preferably culture medium represents less thanabout 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors ornon-protein of interest chemicals.

The nucleotide sequences of the invention include both the naturallyoccurring sequences as well as mutant (variant) forms. Such variantswill continue to possess the desired activity, i.e., either promoteractivity or the activity of the product encoded by the open readingframe of the non-variant nucleotide sequence.

Thus, by “variants” is intended substantially similar sequences. Fornucleotide sequences comprising an open reading frame, variants includethose sequences that, because of the degeneracy of the genetic code,encode the identical amino acid sequence of the native protein.Naturally occurring allelic variants such as these can be identifiedwith the use of well-known molecular biology techniques, as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques. Variant nucleotide sequences also include syntheticallyderived nucleotide sequences, such as those generated, for example, byusing site-directed mutagenesis and for open reading frames, encode thenative protein, as well as those that encode a polypeptide having aminoacid substitutions relative to the native protein. Generally, nucleotidesequence variants of the invention will have at least 40, 50, 60, to70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%,generally at least 80%, e.g., 81%–84%, at least 85%, e.g., 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99%nucleotide sequence identity to the native (wild type or endogenous)nucleotide sequence.

“Conservatively modified variations” of a particular nucleic acidsequence refers to those nucleic acid sequences that encode identical oressentially identical amino acid sequences, or where the nucleic acidsequence does not encode an amino acid sequence, to essentiallyidentical sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenpolypeptide. For instance the codons CGT, CGC, CGA, COG, AGA, and AGGall encode the amino acid arginine. Thus, at every position where anarginine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded protein.Such nucleic acid variations are “silent variations” which are onespecies of “conservatively modified variations.” Every nucleic acidsequence described herein which encodes a polypeptide also describesevery possible silent variation, except where otherwise noted. One ofskill will recognize that each codon in a nucleic acid (except ATG,which is ordinarily the only codon for methionine) can be modified toyield a functionally identical molecule by standard techniques.Accordingly, each “silent variation” of a nucleic acid which encodes apolypeptide is implicit in each described sequence.

The nucleic acid molecules of the invention can be “optimized” forenhanced expression in plants of interest. See, for example, EPA 035472;WO 91/16432; Perlak et al., 1991; and Murray et al., 1989. In thismanner, the open reading frames in genes or gene fragments can besynthesized utilizing plant-preferred codons. See, for example, Campbelland Gowri, 1990 for a discussion of host-preferred codon usage. Thus,the nucleotide sequences can be optimized for expression in any plant.It is recognized that all or any part of the gene sequence may beoptimized or synthetic. That is, synthetic or partially optimizedsequences may also be used. Variant nucleotide sequences and proteinsalso encompass sequences and protein derived from a mutagenic andrecombinogenic procedure such as DNA shuffling. With such a procedure,one or more different coding sequences can be manipulated to create anew polypeptide possessing the desired properties. In this manner,libraries of recombinant polynucleotides are generated from a populationof related sequence polynucleotides comprising sequence regions thathave substantial sequence identity and can be homologously recombined invitro or in vivo. Strategies for such DNA shuffling are known in theart. See, for example, Stemmer, 1994; Stemmer, 1994; Crameri et al.,1997; Moore et al., 1997; Zhang et al., 1997; Crameri et al., 1998; andU.S. Pat. Nos. 5,605,793 and 5,837,458.

By “variant” polypeptide is intended a polypeptide derived from thenative protein by deletion (so-called truncation) or addition of one ormore amino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Such variants may resultfrom, for example, genetic polymorphism or from human manipulation.Methods for such manipulations are generally known in the art.

Thus, the polypeptides may be altered in various ways including aminoacid substitutions, deletions, truncations, and insertions. Methods forsuch manipulations are generally known in the art. For example, aminoacid sequence variants of the polypeptides can be prepared by mutationsin the DNA. Methods for mutagenesis and nucleotide sequence alterationsare well known in the art. See, for example, Kunkel, 1985; Kunkel etal., 1987; U.S. Pat. No. 4,873,192; Walker and Gaastra, 1983 and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978).Conservative substitutions, such as exchanging one amino acid withanother having similar properties, are preferred.

Individual substitutions deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids(typically less than 5%, more typically less than 1%) in an encodedsequence are “conservatively modified variations,” where the alterationsresult in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following five groupseach contain amino acids that are conservative substitutions for oneanother: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L),Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan(W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine I,Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E),Asparagine (N), Glutamine (Q). See also, Creighton, 1984. In addition,individual substitutions, deletions or additions which alter, add ordelete a single amino acid or a small percentage of amino acids in anencoded sequence are also “conservatively modified variations.”

“Expression cassette” as used herein means a DNA sequence capable ofdirecting expression of a particular nucleotide sequence in anappropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest which is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The coding region usually codesfor a protein of interest but may also code for a functional RNA ofinterest, for example antisense RNA or a nontranslated RNA, in the senseor antisense direction. The expression cassette comprising thenucleotide sequence of interest may be chimeric, meaning that at leastone of its components is heterologous with respect to at least one ofits other components. The expression cassette may also be one which isnaturally occurring but has been obtained in a recombinant form usefulfor heterologous expression. The expression of the nucleotide sequencein the expression cassette may be under the control of a constitutivepromoter or of an inducible promoter which initiates transcription onlywhen the host cell is exposed to some particular external stimulus. Inthe case of a multicellular organism, the promoter can also be specificto a particular tissue or organ or stage of development.

“Vector” is defined to include, inter alia, any plasmid, cosmid, phageor Agrobactcrium binary vector in double or single stranded linear orcircular form which may or may not be self transmissible or mobilizable,and which can transform prokaryotic or eukaryotic host either byintegration into the cellular genome or exist extrachromosomally (e.g.autonomous replicating plasmid with an origin of replication).

Specifically included are shuttle vectors by which is meant a DNAvehicle capable, naturally or by design, of replication in two differenthost organisms, which may be selected from actinomycetes and relatedspecies, bacteria and eukaryotic (e.g. higher plant, mammalian, yeast orfungal cells).

Preferably the nucleic acid in the vector is under the control of, andoperably linked to, an appropriate promoter or other regulatory elementsfor transcription in a host cell such as a microbial, e.g. bacterial, orplant cell. The vector may be a bi-functional expression vector whichfunctions in multiple hosts. In the case of genomic DNA, this maycontain its own promoter or other regulatory elements and in the case ofcDNA this may be under the control of an appropriate promoter or otherregulatory elements for expression in the host cell.

“Cloning vectors” typically contain one or a small number of restrictionendonuclease recognition sites at which foreign DNA sequences can beinserted in a determinable fashion without loss of essential biologicalfunction of the vector, as well as a marker gene that is suitable foruse in the identification and selection or cells transformed with thecloning vector. Marker genes typically include genes that providetetracycline resistance, hygromycin resistance or ampicillin resistance.

A “transgenic plant” is a plant having one or more plant cells thatcontain an expression vector.

“Plant tissue” includes differentiated and undifferentiated tissues orplants, including but not limited to roots, stems, shoots, leaves,pollen, seeds, tumor tissue and various forms of cells and culture suchas single cells, protoplast, embryos, and callus tissue. The planttissue may be in plants or in organ, tissue or cell culture.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm. Preferred,non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller, 1988; the local homology algorithm of Smith et al.1981; the homology alignment algorithm of Needleman and Wunsch 1970; thesearch-for-similarity-method of Pearson and Lipman 1988; the algorithmof Karlin and Altschul, 1990, modified as in Karlin and Altschul, 1993.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.1988; Higgins et al. 1989; Corpet et al. 1988; Huang et al. 1992; andPearson et al. 1994. The ALIGN program is based on the algorithm ofMyers and Miller, supra. The BLAST programs of Altschul et al., 1990,are based on the algorithm of Karlin and Altschul supra.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information on the World Wide Web atncbi.nlm.nih.gov/). This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., 1990). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when thecumulative alignment score falls off by the quantity X from its maximumachieved value, the cumulative score goes to zero or below due to theaccumulation of one or more negative-scoring residue alignments, or theend of either sequence is reached.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993). One measure ofsimilarity provided by the BLAST algorithm is the smallest sumprobability (P(N)), which provides an indication of the probability bywhich a match between two nucleotide or amino acid sequences would occurby chance. For example, a test nucleic acid sequence is consideredsimilar to a reference sequence if the smallest sum probability in acomparison of the test nucleic acid sequence to the reference nucleicacid sequence is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized as described in Altschul et al. 1997.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al., supra. When utilizing BLAST, Gapped BLAST,PSIBLAST, the default parameters of the respective programs (e.g. BLASTNfor nucleotide sequences, BLASTX for proteins) can be used. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989). Alignmentmay also be performed manually by inspection.

For purposes of the present invention, comparison of nucleotidesequences for determination of percent sequence identity to the promotersequences disclosed herein is preferably made using the BlastN program(version 1.4.7 or later) with its default parameters or any equivalentprogram. By “equivalent program” is intended any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide or amino acid residue matches and anidentical percent sequence identity when compared to the correspondingalignment generated by the preferred program.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%,91%, 92%, 93%, or 94%, and most preferably at least 95%, 96%, 97%, 98%,or 99% sequence identity, compared to a reference sequence using one ofthe alignment programs described using standard parameters. One of skillin the art will recognize that these values can be appropriatelyadjusted to determine corresponding identity of proteins encoded by twonucleotide sequences by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning, and the like. Substantialidentity of amino acid sequences for these purposes normally meanssequence identity of at least 70%, more is preferably at least 80%, 90%,and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions(see below). Generally, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. However, stringentconditions encompass temperatures in the range of about 1° C. to about20° C., depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is when thepolypeptide encoded by the first nucleic acid is immunologically crossreactive with the polypeptide encoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%,92%, 93%, or 94%, or even more preferably, 95%, 96%, 97%, 98% or 99%,sequence identity to the reference sequence over a specified comparisonwindow. Preferably, optimal alignment is conducted using the homologyalignment algorithm of Needleman and Wunsch (1970). An indication thattwo peptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

As noted above, another indication that two nucleic acid sequences aresubstantially identical is that the two molecules hybridize to eachother under stringent conditions. The phrase “hybridizing specificallyto” refers to the binding, duplexing, or hybridizing of a molecule onlyto a particular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetnucleic acid sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridization are sequence dependent, andare different under different environmental parameters. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Specificity istypically the function of post-hybridization washes, the criticalfactors being the ionic strength and temperature of the final washsolution. For DNA-DNA hybrids, the T_(m) can be approximated from theequation of Meinkoth and Wahl, 1984; T_(m) 81.5° C.+16.6 (log M)+0.41 (%GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations,% GC is the percentage of guanosine and cytosine nucleotides in the DNA,% form is the percentage of formamide in the hybridization solution, andL is the length of the hybrid in base pairs. T_(m) is reduced by about1° C. for each 1% of mismatching; thus, T_(m), hybridization, and/orwash conditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point 1 forthe specific sequence and its complement at a defined ionic strength andpH. However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point 1;moderately stringent conditions can utilize a hybridization and/or washat 6, 7, 8, 9, or 10° C. lower than the thermal melting point 1; lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15, or 20° C. lower than the thermal melting point I. Using theequation, hybridization and wash compositions, and desired T, those ofordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a T of less than 45° C.(aqueous solution) or 32° C. (formamide solution), it is preferred toincrease the SSC concentration so that a higher temperature can be used.An extensive guide to the hybridization of nucleic acids is found inTijssen, 1993. Generally, highly stringent hybridization and washconditions are selected to be about 5° C. lower than the thermal meltingpoint T_(m) for the specific sequence at a defined ionic strength andpH.

An example of highly stringent wash conditions is 0.15 M NaCl at 72° C.for about 15 minutes. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook, infra, for adescription of SSC buffer). Often, a high stringency wash is preceded bya low stringency wash to remove background probe signal. An examplemedium stringency wash for a duplex of, e.g., more than 100 nucleotides,is 1×SSC at 45° C. for 15 minutes. An example low stringency wash for aduplex of, e.g., more than 100 nucleotides, is 4–6×SSC at 40° C. for 15minutes. For short probes (e.g., about 10 to 50 nucleotides), stringentconditions typically involve salt concentrations of less than about 1.5M, more preferably about 0.01 to 1.0 M, Na ion concentration (or othersalts) at pH 7.0 to 8.3, and the temperature is typically at least about30° C. and at least about 60° C. for long robes (e.g., >50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Nucleic acids that do not hybridize to each other understringent conditions are still substantially identical if the proteinsthat they encode are substantially identical. This occurs, e.g., when acopy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

Very stringent conditions are selected to be equal to the T_(m) for aparticular probe. An example of stringent conditions for hybridizationof complementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formamide,e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.1×SSC at 60 to 65° C. Exemplary low stringency conditionsinclude hybridization with a buffer solution of 30 to 35% formamide, 1 MNaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C.Exemplary moderate stringency conditions include hybridization in 40 to45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSCat 55 to 60° C.

The following are examples of sets of hybridization/wash conditions thatmay be used to clone orthologous nucleotide sequences that aresubstantially identical to reference nucleotide sequences of the presentinvention: a reference nucleotide sequence preferably hybridizes to thereference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50C, moredesirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirably stillin 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C.with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC,0.1% SDS at 65° C.

“DNA shuffling” is a method to introduce mutations or rearrangements,preferably randomly, in a DNA molecule or to generate exchanges of DNAsequences between two or more DNA molecules, preferably randomly. TheDNA molecule resulting from DNA shuffling is a shuffled DNA moleculethat is a non-naturally occurring DNA molecule derived from at least onetemplate DNA molecule. The shuffled DNA preferably encodes a variantpolypeptide modified with respect to the polypeptide encoded by thetemplate DNA, and may have an altered biological activity with respectto the polypeptide encoded by the template DNA.

“Recombinant DNA molecule’ is a combination of DNA sequences that arejoined together using recombinant DNA technology and procedures used tojoin together DNA sequences as described, for example, in Sambrook etal., 1989.

The word “plant” refers to any plant, particularly to seed plant, and“plant cell” is a structural and physiological unit of the plant, whichcomprises a cell wall but may also refer to a protoplast. The plant cellmay be in form of an isolated single cell or a cultured cell, or as apart of higher organized unit such as, for example, a plant tissue, or aplant organ.

“Significant increase” is an increase that is larger than the margin oferror inherent in the measurement technique, preferably an increase byabout 2-fold or greater.

“Significantly less” means that the decrease is larger than the marginof error inherent in the measurement technique, preferably a decrease byabout 2-fold or greater.

As used herein, the terms “drought resistance” and “drought tolerance”are used to refer to a plant's increased resistance or tolerance tostress induced by or in response to a reduction in water availability,as compared to normal circumstances, and the ability of the plant tofunction and survive in lower-water environments, and perform in arelatively superior manner. For example, the expression of an isolatedand purified DNA segment encoding a gene product that alters, e.g.,disrupts, inhibits or eliminates, ABA signal transduction in a plantlikely regulates the activity of guard cell stomata and impartsprotection to the plant against drought.

II. The Polynucleotides of the Invention and the Polypeptides EncodedThereby

This invention relates to an isolated plant, e.g., Oryza sativa,polynucleotide, i.e., a nucleic acid molecule, sequence or segment(fragment), which encodes a monocot cap binding protein 80. Thepolynucleotide can, for example, be used to prepare constructs whichoverexpress the encoded protein or a variant thereof, to prepareantisense constructs, to prepare constructs to knock out the expressionof at least one genomic copy of the gene, or to prepare constructscomprising a ribozyme or RNAi specific for cap binding protein 80.

Sources for the polynucleotide of the invention include dicots andmonocots, including but not limited to, corn (Zea mays), Brassica sp.(e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago saliva), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee(Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables, ornamentals, and conifers; duckweed (Lemna, see WO 00/07210,which includes members of the family Lemnaceae.

There are known four genera and 34 species of duckweed as follows: genusLemna (L. aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L.japonica, L. minor, L. miniscula, L. obscura, L. perpusilla, L. tenera,L. trisulca, L. turionifera, L. valdiviana); genus Spirodela (S.intermedia, S. polyrrhiza, S. punctata); genus Woffia (Wa. angusia, Wa.arrhiza, Wa. australina, Wa. borealis, Wa. brasiliensis, Wa. columbiana,Wa. elongata, Wa. globosa, Wa. microscopica, Wa. neglecia) and genusWofiella (Wl. caudata, Wl. denticulata, Wl. gladiata, Wl. hyalina, Wl.lingulara, Wl. repunda, Wl. rotunda, and Wl. neotropica). Any othergenera or species of Lemnaceae, if they exist, are also aspects of thepresent invention. Lemna gibba, Lemna minor, and Lemna miniscula arepreferred, with Lemna minor and Lemna miniscula being most preferred.Lemna species can be classified using the taxonomic scheme described byLandolt, Biosystematic Investigation on the Family of Duckweeds: Thefamily of Lemnaceae—A Monograph Study. Geobotanisches Institut ETH,Stiftung Rubel, Zurich (1986)); vegetables including tomatoes(Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans(Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (lathyrusspp.), and members of the genus Cucumis such as cucumber (C. sativus),cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentalsinclude azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea),hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipaspp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation(Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), andchrysanthemum. Conifers that may be employed in practicing the presentinvention include, for example, pines such as loblolly pine (Pinustaeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa),lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata),Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis);Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firssuch as silver fir (Abies amabilis) and balsam fir (Abies balsamea); andcedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis); and leguminous plants.

Leguminous plants include beans and peas. Beans include guar, locustbean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean,fava bean, lentils, chickpea, etc. Legumes include, but are not limitedto, Arachis, e.g., peanuts, Vicia, e.g., crown vetch, hairy vetch,adzuki bean, mung bean, and chickpea, Lupinus, e.g., lupine, trifolium,Phaseolus, e.g., common bean and lima bean, Pisum, e.g., field bean,Melilotus, e.g., clover, Medicago, e.g., alfalfa, Lotus, e.g., trefoil,lens, e.g., lentil, and false indigo. Other sources for thepolynucleotides of the invention include Acacia, aneth, artichoke,arugula, blackberry, canola, cilantro, clementines, escarole,eucalyptus, fennel, grapefruit, honey dew, jicama, kiwifruit, lemon,lime, mushroom, nut, okra, orange, parsley, persimmon, plantain,pomegranate, poplar, radiata pine, radicchio, Southern pine, sweetgum,tangerine, triticale, vine, yams, apple, pear, quince, cherry, apricot,melon, hemp, buckwheat, grape, raspberry, chenopodium, blueberry,nectarine, peach, plum, strawberry, watermelon, eggplant, pepper,cauliflower, Brassica, e.g., broccoli, cabbage, brussel sprouts, onion,carrot, leek, beet, broad bean, celery, radish, pumpkin, endive, gourd,garlic, snapbean, spinach, squash, turnip, asparagus, and zucchini andornamental plants include impatiens, Begonia, Pelargonium, Viola,Cyclamen, Verbena, Vinca, Tagetes, Primula, Saint Paulia, Agertum,Amaranthus, Antihirrhinum, Aquilegia, Cineraria, Clover, Cosmo, Cowpea,Dahlia, Datura, Delphinium, Gerbera, Gladiolus, Gloxinia, Hippeastrum,Mesembryanthemum, Salpiglossos, and Zinnia.

Other vegetable sources (and databases to identify orthologs of theinvention) for the nucleic acid sequences of the invention includeCucurbitaceae, e.g., Cucumis sativus, Cucumis melo, Citrullus lanatus,Cucurbila pepo, Cucurbita maxima, and Cucurbita moschata (see the WorldWide Web at cucurbit.org/, genome.cornell.edu/cgc/ornal.usda.gov/pgdic/Map_proj/); Solanaceae, e.g., Lycopersiconesculentum, Capsiewn annuum, Capsicum frutescens, Solinum melongena,Nicotiana tabacum, Solanum tuberosum, Petunia×hybrida hort. ex E. Vilm.(see, 15×BAC on variety Heinz 1706 order from Clemson Genome center onthe World Wide Web at genome.clemson.edu, 11.6×BAC of L. cheesmanii(originates from J. Giovannoni) available from Clemson genome center onthe World Wide Web at genome.clemson.edu, EST collection from TIGR onthe World Wide Web at tigr.org/tdb/lgi/index.html, EST collection fromClemson Genome Center on the World Wide Web at genome.clemson.edu,esculentum×pennelli peruvianum, potato×tomato, potato and tomato,esculentum×pennelli isozyme and cDNAs, 4×BAC of Petunia hybrida 7984available from Clemson genome center on the World Wide Web atgenome.clemson.edu, genome.cornell.edu/solgenes,ars-genome.cornell.edu/cgi-bin/WebAce/webace?db=solgenes,genome.cornell.edu/tgc/, tgrc.ucdavis.edu,neptune.netimages.com/.about.chile/science.html, andnal.usda.gov/pgdic/Map_proi.; Brassicaceae, e.g., Brassica oleracea L.var. italica, Brassica oleracea L. var. capitata, Brassica rapa,Brassica oleracea L. var. botrytis, Raphanus sativus var. niger, andBrassica napus (see, 12× and 6×BACs on Columbia strain available fromthe following World Wide Web sites: genome.clemson.edu (Clemson genomecenter), res.agr.ca/ecorc/cwmt/crucifer/traits/index.htm,geneous.cit.cornell.edu/cabbage/aboutcab.html,ars-genome.comell.edu/cgi-bin/WebAce/webace?db=brassicadb,ars-genome.comell.edu/cgi-bin/WebAce/webace?db=agr,nal.usda.gov/pgdic/MAP_proj/; Umbelliferae, e.g., Daucus carota,Compositae, e.g., Lactuca sativa, and Helianthus annuus; Chenopodiaceac,e.g., Spinacia oleracea and Beta vulgaris; Leguminosae, e.g., Phaseolusvulgas, Pisum sativum, and Glycine max (see, 4.3×BAC available fromClemson genome center on the World Wide Web at gcnome.clemson.edu, 7.5×and 7.9×BACs available from the following World Wide Web sites:genome.clemson.edu, nal.usda.gov/pgdic/Map_proj/,ars-genome.cornell.edu/cgi-bin/webAce/webace?db=beangenes, orars-genome.comell.edu/cgi-bin/WebAce/webace?db=soybase; Gramineae, e.g.,Zea mays, see, Novartis BACs for Mo 17 and B73 have been donated toClemson Genome Center and are available at the following World Wide Websites: genome.clemson.edu, nal.usda.gov/pgdic/Map_proj/, andagron.missouri.ed-u/mnl/; or Liliaceae, e.g., Allium cepa (see, theWorld Wide Web site nal.usda.gov/pgdi-c/Map_proj/.

Preferred forage and turf grass nucleic acid sources for thepolynucleotides of the invention include alfalfa, orchard grass, tallfescue, perennial ryegrass, creeping bent grass, and redtop. Preferably,the nucleic acid sources are crop plants and in particular cereals,e.g., wheat, rice, corn, oats, barley, and rye, and even more preferablyrice, corn and wheat.

Based on the Oryza nucleotide sequence of the present invention,orthologs may be identified or isolated from the genome of any desiredorganism, preferably from another plant, according to well knowntechniques based on their sequence similarity to the Oryza nucleic acidsequences, e.g., hybridization, PCR or computer generated sequencecomparisons. For example, all or a portion of a particular Oryzanucleotide sequence is used as a probe that selectively hybridizes toother gene sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen source organism. Further, suitable genomic and cDNA libraries maybe prepared from any cell or tissue of an organism. Such techniquesinclude hybridization screening of plated DNA libraries (either plaquesor colonies; see, e.g., Sambrook et al., 1989) and amplification by PCRusing oligonucleotide primers preferably corresponding to sequencedomains conserved among related polypeptide or subsequences of thenucleotide sequences provided herein (see, e.g., Innis et al., 1995).These methods are particularly well suited to the isolation of genesequences from organisms closely related to the organism from which theprobe sequence is derived.

The application of these methods using the Oryza sequences as probes iswell suited for the isolation of gene sequences from any sourceorganism, preferably other plant species. In a PCR approach,oligonucleotide primers can be designed for use in PCR reactions toamplify corresponding DNA sequences from cDNA or genomic DNA extractedfrom any plant of interest. Methods for designing PCR primers and PCRcloning are generally known in the art.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P, or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the sequence of theinvention. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed in Sambrook et al., 1989. In general, sequencesthat hybridize to the sequences disclosed herein will have at least 40%to 50%, about 60% to 70% and even about 80% 85%, 90%, 95% to 98% or moreidentity with the disclosed sequences. That is, the sequence similarityof sequences may range, sharing at least about 40% to 50%, about 60% to70%, and even about 80%, 85%, 90%, 95% to 98% sequence similarity.

The polynucleotides of the invention can also be identified by, forexample, a search of known databases for genes encoding polypeptideshaving a specified amino acid sequence identity. Methods of alignment ofsequences for comparison are well known in the art and are describedhereinabove.

III. DNA Sequences for Transformation

Virtually any DNA composition may be used for delivery to recipientplant cells, e.g., monocotyledonous cells, to ultimately produce fertiletransgenic plants in accordance with the present invention. For example,DNA segments in the form of vectors and plasmids, or linear DNAfragments, in some instances containing only the DNA element to beexpressed in the plant, and the like, may be employed. The constructionof vectors which may be employed in conjunction with the presentinvention will be known to those of skill of the art in light of thepresent disclosure (see, e.g., Sambrook et al., 1989; Gelvin et al.,1990).

Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes), BACs(bacterial artificial chromosomes) and DNA segments for use intransforming such cells will, of course, generally comprise the cDNA,gene or genes which one desires to introduce into the cells. These DNAconstructs can further include structures such as promoters, enhancers,polylinkers, or even regulatory genes as desired. The DNA segment orgene chosen for cellular introduction will often encode a protein whichwill be expressed in the resultant recombinant cells, such as willresult in a screenable or selectable trait and/or which will impart animproved phenotype to the regenerated plant. However, this may notalways be the case, and the present invention also encompassestransgenic plants incorporating non-expressed transgenes.

In certain embodiments, it is contemplated that one may wish to employreplication-competent viral vectors in monocot transformation. Suchvectors include, for example, wheat dwarf virus (WDV) “shuttle” vectors,such as pW1-11 and PW1-GUS (Ugaki et al., 1991). These vectors arecapable of autonomous replication in maize cells as well as E. coli, andas such may provide increased sensitivity for detecting DNA delivered totransgenic cells. A replicating vector may also be useful for deliveryof genes flanked by DNA sequences from transposable elements such as Ac,Ds, or Mu. It has been proposed (Laufs et al., 1990) that transpositionof these elements within the maize genome requires DNA replication. Itis also contemplated that transposable elements would be useful forintroducing DNA fragments lacking elements necessary for selection andmaintenance of the plasmid vector in bacteria, e.g., antibioticresistance genes and origins of DNA replication. It is also proposedthat use of a transposable element such as Ac, Ds, or Mu would activelypromote integration of the desired DNA and hence increase the frequencyof stably transformed cells. The use of a transposable element such asAc, Ds, or Mu may actively promote integration of the DNA of interestand hence increase the frequency of stably transformed cells.Transposable elements may be useful to allow separation of genes ofinterest from elements necessary for selection and maintenance of aplasmid vector in bacteria or selection of a transformant. By use of atransposable element, desirable and undesirable DNA sequences may betransposed apart from each other in the genome, such that throughgenetic segregation in progeny, one may identify plants with either thedesirable or the undesirable DNA sequences.

DNA useful for introduction into plant cells includes that which hasbeen derived or isolated from any source, that may be subsequentlycharacterized as to structure, size and/or function, chemically altered,and later introduced into plants. An example of DNA “derived” from asource, would be a DNA sequence that is identified as a useful fragmentwithin a given organism, and which is then chemically synthesized inessentially pure form. An example of such DNA “isolated” from a sourcewould be a useful DNA sequence that is excised or removed from saidsource by chemical means, e.g., by the use of restriction endonucleases,so that it can be further manipulated, e.g., amplified, for use in theinvention, by the methodology of genetic engineering. Such DNA iscommonly referred to as “recombinant is DNA.”

Therefore useful DNA includes completely synthetic DNA, semi-syntheticDNA, DNA isolated from biological sources, and DNA derived fromintroduced RNA. Generally, the introduced DNA is not originally residentin the plant genotype which is the recipient of the DNA, but it iswithin the scope of the invention to isolate a gene from a given plantgenotype, and to subsequently introduce multiple copies of the gene intothe same genotype, e.g., to enhance production of a given gene productsuch as a storage protein or a protein that confers tolerance orresistance to water deficit.

The introduced DNA includes but is not limited to, DNA from plant genes,and non-plant genes such as those from bacteria, yeasts, animals orviruses. The introduced DNA can include modified genes, portions ofgenes, or chimeric genes, including genes from the same or differentmaize genotype. The term “chimeric gene” or “chimeric DNA” is defined asa gene or DNA sequence or segment comprising at least two DNA sequencesor segments from species which do not combine DNA under naturalconditions, or which DNA sequences or segments are positioned or linkedin a manner which does not normally occur in the native genome ofuntransformed plant.

The introduced DNA used for transformation herein may be circular orlinear, double-stranded or single-stranded. Generally, the DNA is in theform of chimeric DNA, such as plasmid DNA, that can also contain codingregions flanked by regulatory sequences which promote the expression ofthe recombinant DNA present in the resultant plant. For example, the DNAmay itself comprise or consist of a promoter that is active in a plantwhich is derived from a source other than that plant, or may utilize apromoter already present in a plant genotype that is the transformationtarget.

Generally, the introduced DNA will be relatively small, i.e., less thanabout 30 kb to minimize any susceptibility to physical, chemical, orenzymatic degradation which is known to increase as the size of the DNAincreases. As noted above, the number of proteins, RNA transcripts ormixtures thereof which is introduced into the plant genome is preferablypreselected and defined, e.g., from one to about 5–10 such products ofthe introduced DNA may be formed.

Two principal methods for the control of expression are known, viz.:overexpression and underexpression. Overexpression can be achieved byinsertion of one or more than one extra copy of the selected gene. Itis, however, not unknown for plants or their progeny, originallytransformed with one or more than one extra copy of a nucleotidesequence, to exhibit the effects of underexpression as well asoverexpression. For underexpression there are two principle methodswhich are commonly referred to in the art as “antisense downregulation”and “sense downregulation” (sense downregulation is also referred to as“cosuppression”). Generically these processes are referred to as “genesilencing”. Both of these methods lead to an inhibition of expression ofthe target gene.

Obtaining sufficient levels of transgene expression in the appropriateplant tissues is an important aspect in the production of geneticallyengineered crops. Expression of heterologous DNA sequences in a planthost is dependent upon the presence of an operably linked promoter thatis functional within the plant host. Choice of the promoter sequencewill determine when and where within the organism the heterologous DNAsequence is expressed.

Furthermore, it is contemplated that promoters combining elements frommore than one promoter may be useful. For example, U.S. Pat. No.5,491,288 discloses combining a Cauliflower Mosaic Virus promoter with ahistone promoter. Thus, the elements from the promoters disclosed hereinmay be combined with elements from other promoters.

Promoters which are useful for plant transgene expression include thosethat are inducible, viral, synthetic, constitutive (Odell et al., 1985),temporally regulated, spatially regulated, tissue-specific, andspatio-temporally regulated.

Where expression in specific tissues or organs is desired,tissue-specific promoters may be used. In contrast, where geneexpression in response to a stimulus is desired, inducible promoters arethe regulatory elements of choice. Where continuous expression isdesired throughout the cells of a plant, constitutive promoters areutilized. Additional regulatory sequences upstream and/or downstreamfrom the core promoter sequence may be included in expression constructsof transformation vectors to bring about varying levels of expression ofheterologous nucleotide sequences in a transgenic plant.

A. Transcription Regulatory Sequences

1. Promoters

The choice of promoter will vary depending on the temporal and spatialrequirements for expression, and also depending on the target species.In some cases, expression in multiple tissues is desirable. While inothers, tissue-specific, e.g., leaf-specific, seed-specific,petal-specific, anther-specific, or pith-specific, expression isdesirable. Although many promoters from dicotyledons have been shown tobe operational in monocotyledons and vice versa, ideally dicotyledonouspromoters are selected for expression in dicotyledons, andmonocotyledonous promoters for expression in monocotyledons: However,there is no restriction to the provenance of selected promoters; it issufficient that they are operational in driving the expression of thenucleotide sequences in the desired cell.

These promoters include, but are not limited to, constitutive,inducible, temporally regulated, developmentally regulated,spatially-regulated, chemically regulated, stress-responsive,tissue-specific, viral and synthetic promoters. Promoter sequences areknown to be strong or weak. A strong promoter provides for a high levelof gene expression, whereas a weak promoter provides for a very lowlevel of gene expression. An inducible promoter is a promoter thatprovides for the turning on and off of gene expression in response to anexogenously added agent, or to an environmental or developmentalstimulus. A bacterial promoter such as the P_(lac) promoter can beinduced to varying levels of gene expression depending on the level ofisothiopropylgalactoside added to the transformed bacterial cells. Anisolated promoter sequence that is a strong promoter for heterologousnucleic acid is advantageous because it provides for a sufficient levelof gene expression to allow for easy detection and selection oftransformed cells and provides for a high level of gene expression whendesired.

Within a plant promoter region there are several domains that arenecessary for full function of the promoter. The first of these domainslies immediately upstream of the structural gene and forms the “corepromoter region” containing consensus sequences, normally 70 base pairsimmediately upstream of the gene. The core promoter region contains thecharacteristic CAAT and TATA boxes plus surrounding sequences, andrepresents a transcription initiation sequence that defines thetranscription start point for the structural gene.

The presence of the core promoter region defines a sequence as being apromoter: if the region is absent, the promoter is non-functional.Furthermore, the core promoter region is insufficient to provide fullpromoter activity. A series of regulatory sequences upstream of the coreconstitute the remainder of the promoter. The regulatory sequencesdetermine expression level, the spatial and temporal pattern ofexpression and, for an important subset of promoters, expression underinductive conditions (regulation by external factors such as light,temperature, chemicals, hormones).

A range of naturally-occurring promoters are known to be operative inplants and have been used to drive the expression of heterologous (bothforeign and endogenous) genes in plants: for example, the constitutive35S cauliflower mosaic virus (CaMV) promoter, the ripening-enhancedtomato polygalacturonase promoter (Bird et al., 1988), the E8 promoter(Diekman & Fischer, 1988) and the fruit specific 2A1 promoter (Pear etal., 1989) and many others, e.g., U2 and U5 mRNA promoters from maize,the promoter from alcohol dehydrogenase, the Z4 promoter from a geneencoding the Z422 kD zein protein, the Z10 promoter from a gene encodinga 10 kD zein protein, a Z27 promoter from a gene encoding a 27 kD zeinprotein, the A20 promoter from the gene encoding a 19 kcD-zein protein,inducible promoters, such as the light inducible promoter derived fromthe pea rbcS gene and the actin promoter from rice, e.g., the actin 2promoter (WO 00/70067); seed specific promoters, such as the phaseolinpromoter from beans, may also be used. The nucleotide sequences of thisinvention can also be expressed under the regulation of promoters thatare chemically regulated. This enables the nucleic acid sequence orencoded polypeptide to be synthesized only when the crop plants aretreated with the inducing chemicals. Chemical induction of geneexpression is detailed in EP 0 332 104 (to Ciba-Geigy) and U.S. Pat. No.5,614,395. A preferred promoter for chemical induction is the tobaccoPR-1a promoter.

Examples of some constitutive promoters which have been describedinclude the rice actin 1 (Wang et al., 1992; U.S. Pat. No. 5,641,876),CaMV 35S (Odell et al., 1985), CaMV 19S (Lawton et al., 1987), nos, Adh,sucrose synthase; and the ubiquitin promoters.

Examples of tissue specific promoters which have been described includethe lectin (Vodkin, 1983; Lindstrom et al., 1990) corn alcoholdehydrogenase 1 (Vogel et al., 1989; Dennis et al., 1984), corn lightharvesting complex (Simpson, 1986; Bunsal et al., 1992), corn heat shockprotein (Odell et al., 1985), pea small subunit RuBP carboxylase(Poulsen et al., 1986), Ti plasmid mannopine synthase (Langridge et al.,1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petuniachalcone isomerase (vanTunen et al., 1988), bean glycine rich protein 1(Keller et al., 1989), truncated CaMV 35s (Odell et al., 1985), potatopatatin (Wenzler et al., 1989), root cell (Yamamoto et al., 1990), maizezein (Reina et al., 1990; Kriz et al., 1987; Wandelt et al., 1989;Langridge et al., 1983; Reina et al., 1990), globulin-1 (Belanger etal., 1991), α-tubulin, cab (Sullivan et al., 1989), PEPCase (Hudspeth &Grula, 1989), R gene complex-associated promoters (Chandler et al.,1989), histone, and chalcone synthase promoters (Franken et al., 1991).Tissue specific enhancers are described in Fromm et al. (1989).

Inducible promoters that have been described include the ABA- andturgor-inducible promoters, the promoter of the auxin-binding proteingene (Schwob et al., 1993), the UDP glucose flavonoidglycosyl-transferase gene promoter (Ralston et al., 1988), the MPIproteinase inhibitor promoter (Cordero et al., 1994), and theglyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al.,1995; Quigley et al., 1989; Martinez et al., 1989).

Several other tissue-specific regulated genes and/or promoters have beenreported in plants. These include genes encoding the seed storageproteins (such as napin, cruciferin, beta-conglycinin, and phaseolin)zein or oil body proteins (such as oleosin), or genes involved in fattyacid biosynthesis (including acyl carrier protein, stearoyl-ACPdesaturase. And fatty acid desaturases (fad 2-1)), and other genesexpressed during embryo development (such as Bce4, see, for example, EP255378 and Kridl et al., 1991). Particularly useful for seed-specificexpression is the pea vicilin promoter (Czako et al., 1992). (See alsoU.S. Pat. No. 5,625,136, herein incorporated by reference.) Other usefulpromoters for expression in mature leaves are those that are switched onat the onset of senescence, such as the SAG promoter from Arabidopsis(Gan et al., 1995). .

A class of fruit-specific promoters expressed at or during antithesisthrough fruit development, at least until the beginning of ripening, isdiscussed in U.S. Pat. No. 4,943,674. cDNA clones that arepreferentially expressed in cotton fiber have been isolated (John etal., 1992). cDNA clones from tomato displaying differential expressionduring fruit development have been isolated and characterized (Manssonet al., 1985, Slater et al., 1985). The promoter for polygalacturonasegene is active in fruit ripening. The polygalacturonase gene isdescribed in U.S. Pat. Nos. 4,535,060, 4,769,061, 4,801,590, and5,107,065, which disclosures are incorporated herein by reference.

Other examples of tissue-specific promoters include those that directexpression in leaf cells following damage to the leaf (for example, fromchewing insects), in tubers (for example, patatin gene promoter), and infiber cells (an example of a developmentally-regulated fiber cellprotein is E6 (John et al., 1992). The E6 gene is most active in fiber,although low levels of transcripts are found in leaf, ovule and flower.

The tissue-specificity of some “tissue-specific” promoters may not beabsolute and may be tested by one skilled in the art using thediphtheria toxin sequence. One can also achieve tissue-specificexpression with “leaky” expression by a combination of differenttissue-specific promoters (Beals et al., 1997). Other tissue-specificpromoters can be isolated by one skilled in the art (see U.S. Pat. No.5,589,379). Several inducible promoters (“gene switches”) have beenreported. Many are described in the review by Gatz (1996) and Gatz(1997). These include tetracycline repressor system, Lac repressorsystem, copper-inducible systems, salicylate-inducible systems (such asthe PR1a system), glucocorticoid (Aoyama et al., 1997) andecdysone-inducible systems. Also included are the benzene sulphonamide(U.S. Pat. No. 5,364,780) and alcohol-(WO 97/06269 and WO 97/06268)inducible systems and glutathione S-transferase promoters. Other studieshave focused on genes inducibly regulated in response to environmentalstress or stimuli such as increased salinity. Drought, pathogen andwounding. (Graham et al., 1985; Graham et al., 1985, Smith et al.,1986). Accumulation of metallocarboxypeptidase-inhibitor protein hasbeen reported in leaves of wounded potato plants (Graham et al., 1981).Other plant genes have been reported to be induced methyl jasmonate,elicitors, heat-shock, anaerobic stress, or herbicide safeners.

Regulated expression of the chimeric transacting viral replicationprotein can be further regulated by other genetic strategies. Forexample, Cre-mediated gene activation as described by Odell et al. 1990.Thus, a DNA fragment containing 3′ regulatory sequence bound by loxsites between the promoter and the replication protein coding sequencethat blocks the expression of a chimeric replication gene from thepromoter can be removed by Cre-mediated excision and result in theexpression of the trans-acting replication gene. In this case, thechimeric Cre gene, the chimeric trans-acting replication gene, or bothcan be under the control of tissue- and developmental-specific orinducible promoters. An alternate genetic strategy is the use of tRNAsuppressor gene. For example, the regulated expression of a tRNAsuppressor gene can conditionally control expression of a trans-actingreplication protein coding sequence containing an appropriatetermination codon as described by Ulmasov et al. 1997. Again, either thechimeric tRNA suppressor gene, the chimeric transacting replicationgene, or both can be under the control of tissue- anddevelopmental-specific or inducible promoters.

Frequently it is desirable to have continuous or inducible expression ofa DNA sequence throughout the cells of an organism in atissue-independent manner. For example, increased resistance of a plantto infection by soil- and airborne-pathogens might be accomplished bygenetic manipulation of the plant's genome to comprise a continuouspromoter operably linked to a heterologous pathogen-resistance gene suchthat pathogen-resistance proteins are continuously expressed throughoutthe plant's tissues.

Alternatively, it might be desirable to inhibit expression of a nativeDNA sequence within a plant's tissues to achieve a desired phenotype. Inthis case, such inhibition might be accomplished with transformation ofthe plant to comprise a constitutive, tissue-independent promoteroperably linked to an antisense nucleotide sequence, such thatconstitutive expression of the antisense sequence produces an RNAtranscript that interferes with translation of the mRNA of the nativeDNA sequence.

To define a minimal promoter region, a DNA segment representing thepromoter region is removed from the 5′ region of the gene of interestand operably linked to the coding sequence of a marker (reporter) geneby recombinant DNA techniques well known to the art. The reporter geneis operably linked downstream of the promoter, so that transcriptsinitiating at the promoter proceed through the reporter gene. Reportergenes generally encode proteins which are easily measured, including,but not limited to, chloramphenicol acetyl transferase (CAT),beta-glucuronidase (GUS), green fluorescent protein (GFP),beta-galactosidase (beta-GAL), and luciferase.

The construct containing the reporter gene under the control of thepromoter is then introduced into an appropriate cell type bytransfection techniques well known to the art. To assay for the reporterprotein, cell lysates are prepared and appropriate assays, which arewell known in the art, for the reporter protein are performed. Forexample, if CAT were the reporter gene of choice, the lysates from cellstransfected with constructs containing CAT under the control of apromoter under study are mixed with isotopically labeled chloramphenicoland acetyl-coenzyme A (acetyl-CoA). The CAT enzyme transfers the acetylgroup from acetyl-CoA to the 2- or 3-position of chloramphenicol. Thereaction is monitored by thin-layer chromatography, which separatesacetylated chloramphenicol from unreacted material. The reactionproducts are then visualized by autoradiography.

The level of enzyme activity corresponds to the amount of enzyme thatwas made, which in turn reveals the level of expression from thepromoter of interest. This level of expression can be compared to otherpromoters to determine the relative strength of the promoter understudy. In order to be sure that the level of expression is determined bythe promoter, rather than by the stability of the mRNA, the level of thereporter mRNA can be measured directly, such as by Northern blotanalysis.

Once activity is detected, mutational and/or deletional analyses may beemployed to determine the minimal region and/or sequences required toinitiate transcription. Thus, sequences can be deleted at the 5′ end ofthe promoter region and/or at the 3′ end of the promoter region, andnucleotide substitutions introduced. These constructs are thenintroduced to cells and their activity determined.

In one embodiment, the promoter may be a gamma zein promoter, an oleosinole16 promoter, a globulinI promoter, an actin I promoter, an actin clpromoter, a sucrose synthetase promoter, an INOPS promoter, an EXM5promoter, a globulin2 promoter, a b-32, ADPG-pyrophosphorylase promoter,an LtpI promoter, an Ltp2 promoter, an oleosin ole17 promoter, anoleosin ole18 promoter, an actin 2 promoter, a pollen-specific proteinpromoter, a pollen-specific pectate lyase promoter, an anther-specificprotein promoter (Huffman), an anther-specific gene RTS2 promoter, apollen-specific gene promoter, a tapetum-specific gene promoter,tapetum-specific gene RAB24 promoter, a anthranilate synthase alphasubunit promoter, an alpha zein promoter, an anthranilate synthase betasubunit promoter. a dihydrodipicolinate synthase promoter, a Thilpromoter, an alcohol dehydrogenase promoter, a cab binding proteinpromoter, an H3C4 promoter, a RUBISCO SS starch branching enzymepromoter, an ACCase promoter, an actin3 promoter, an actin7 promoter, aregulatory protein GF14-12 promoter, a ribosomal protein L9 promoter, acellulose biosynthetic enzyme promoter, an S-adenosyl-L-homocysteinehydrolase promoter, a superoxide dismutase promoter, a C-kinase receptorpromoter, a phosphoglycerate mutase promoter, a root-specific RCc3 mRNApromoter, a glucose-6 phosphate isomerase promoter, apyrophosphate-fructose 6-phosphatelphosphotransferase promoter, anubiquitin promoter, a beta-ketoacyl-ACP synthase promoter, a 33 kDaphotosystem 11 promoter, an oxygen evolving protein promoter, a 69 kDavacuolar ATPase subunit promoter, a metallothionein-like proteinpromoter, a glyceraldehyde-3-phosphate dehydrogenase promoter, an ABA-and ripening-inducible-like protein promoter, a phenylalanine ammonialyase promoter, an adenosine triphosphatase Sadenosyl-L-homocysteinehydrolase promoter, an a-tubulin promoter, a cab promoter, a PEPCasepromoter, an R gene promoter, a lectin promoter, a light harvestingcomplex promoter, a heat shock protein promoter, a chalcone synthasepromoter, a zein promoter, a globulin-1 promoter, an ABA promoter, anauxin-binding protein promoter, a UDP glucose flavonoidglycosyl-transferase gene promoter, an NTI promoter, an actin promoter,an opaque 2 promoter, a b70 promoter, an oleosin promoter, a CaMV 35Spromoter, a CaMV 19S promoter, a histone promoter, a turgor-induciblepromoter, a pea small subunit RuBP carboxylase promoter, a Ti plasmidmannopine synthase promoter, Ti plasmid nopaline synthase promoter, apetunia chalcone isomerase promoter, a bean glycine rich protein Ipromoter, a CaMV 35S transcript promoter, a potato patatin promoter, ora S-E9 small subunit RuBP carboxylase promoter.

2. Other Regulatory Elements

In addition to promoters, a variety of 5N and 3N transcriptionalregulatory sequences are also available for use in the presentinvention. Transcriptional terminators are responsible for thetermination of transcription and correct mRNA polyadenylation. The 3Nnontranslated regulatory DNA sequence preferably includes from about 50to about 1,000, more preferably about 100 to about 1,000, nucleotidebase pairs and contains plant transcriptional and translationaltermination sequences. Appropriate transcriptional terminators and thosewhich are known to function in plants include the CAMV 35S terminator,the tml terminator, the nopaline synthase terminator, the pea rbcS E9terminator, the terminator for the T7 transcript from the octopinesynthase gene of Agrobacterium tumefaciens, and the 3N end of theprotease inhibitor I or II genes from potato or tomato, although other3N elements known to those of skill in the art can also be employed.Alternatively, one also could use a gamma coixin, oleosin 3 or otherterminator from the genus Coix.

Preferred 3′ elements include those from the nopaline synthase gene ofAgrobacterium tumefaciens (Bevan et al., 1983), the terminator for theT7 transcript from the octopine synthase gene of Agrobacteriumtumefaciens, and the 3′ end of the protease inhibitor 1 or 11 genes frompotato or tomato.

As the DNA sequence between the transcription initiation site and thestart of the coding sequence, i.e., the untranslated leader sequence,can influence gene expression, one may also wish to employ a particularleader sequence. Preferred leader sequences are contemplated to includethose which include sequences predicted to direct optimum expression ofthe attached gene, i.e., to include a preferred consensus leadersequence which may increase or maintain mRNA stability and preventinappropriate initiation of translation. The choice of such sequenceswill be known to those of skill in the art in light of the presentdisclosure. Sequences that are derived from genes that are highlyexpressed in plants will be most preferred.

Other sequences that have been found to enhance gene expression intransgenic plants include intron sequences (e.g.; from Adh1, bronze1,actin1, actin 2 (WO 00/760067), or the sucrose synthase intron) andviral leader sequences (e.g., from TMV, MCMV and AMV). For example, anumber of non-translated leader sequences derived from viruses are knownto enhance expression. Specifically, leader sequences from TobaccoMosaic Virus (TMV), Maize Chlorotic Mottle Virus (MCMV), and AlfalfaMosaic Virus (AMV) have been shown to be effective in enhancingexpression (e.g., Gallie et al., 1987; Skuzeski et al., 1990). Otherleaders known in the art include but are not limited to: Picomavirusleaders, for example, EMCV leader (Encephalomyocarditis 5 noncodingregion) (Elroy-Stein et al., 1989); Potyvirus leaders, for example, TEVleader (Tobacco Etch Virus); MDMV leader (Maize Dwarf Mosaic Virus);Human immunoglobulin heavy-chain binding protein (BiP) leader, (Macejaket al., 1991); Untranslated leader from the coat protein mRNA of alfalfamosaic virus (AMV RNA 4), (Jobling et al., 1987; Tobacco mosaic virusleader (TMV), (Gallie et al., 1989; and Maize Chlorotic Mottle Virusleader (MCMV) (Lommel et al., 1991. See also, Della-Cioppa et al., 1987.

Regulatory elements such as Adh intron 1 (Callis et al., 1987), sucrosesynthase intron (Vasil et al., 1989) or TMV omega element (Gallie, etal., 1989), may further be included where desired.

Examples of enhancers include elements from the CAMV 35S promoter,octopine synthase genes (Ellis et al., 1987), the rice actin I gene, themaize alcohol dehydrogenase gene (Callis et al., 1987), the maizeshrunken I gene (Vasil et al., 1989), TMV Omega element (Gallie et al.,1989) and promoters from non-plant eukaryotes (e.g. yeast; Ma et al.,1988).

Vectors for use in accordance with the present invention may beconstructed to include the ocs enhancer element. This element was firstidentified as a 16 bp palindromic enhancer from the octopine synthase(ocs) gene of ultilane (Ellis et al., 1987), and is present in at least10 other promoters (Bouchez et al., 1989). The use of an enhancerelement, such as the ocs element and particularly multiple copies of theelement, will act to increase the level of transcription from adjacentpromoters when applied in the context of monocot transformation.

Ultimately, the most desirable DNA segments for introduction into forexample a monocot genome may be homologous genes or gene families whichencode a desired trait (e.g., increased yield per acre) and which areintroduced under the control of novel promoters or enhancers, etc., orperhaps even homologous or tissue specific (e.g., root-, collar/sheath-,whorl-, stalk-, earshank-, kernel- or leaf-specific) promoters orcontrol elements. Indeed, it is envisioned that a particular use of thepresent invention will be the targeting of a gene in a constitutivemanner or a root-specific manner. For example, insect resistant genesmay be expressed specifically in the whorl and collar/sheath tissueswhich are targets for the first and second broods, respectively, of ECB.Likewise, genes encoding proteins with particular activity againstrootworm may be targeted directly to root tissues.

Vectors for use in tissue-specific targeting of genes in transgenicplants will typically include tissue-specific promoters and may alsoinclude other tissue-specific control elements such as enhancersequences. Promoters which direct specific or enhanced expression incertain plant tissues will be known to those of skill in the art inlight of the present disclosure. These include, for example, the rbcSpromoter, specific for green tissue; the ocs, nos and mas promoterswhich have higher activity in roots or wounded leaf tissue; a truncated(−90 to +8) 35S promoter which directs enhanced expression in roots, analpha-tubulin gene that directs expression in roots and promotersderived from zein storage protein genes which direct expression inendosperm. It is particularly contemplated that one may advantageouslyuse the 16 bp ocs enhancer element from the octopine synthase (ocs) gene(Ellis et al., 1987; Bouchez et al., 1989), especially when present inmultiple copies, to achieve enhanced expression in roots.

Tissue specific expression may be functionally accomplished byintroducing a constitutively expressed gene (all tissues) in combinationwith an antisense gene that is expressed only in those tissues where thegene product is not desired. For example, a gene coding for the crystaltoxin protein from B. thuringiensis (Bt) may be introduced such that itis expressed in all tissues using the 35S promoter from CauliflowerMosaic Virus. Expression of an antisense transcript of the Bt gene in amaize kernel, using for example a zein promoter, would preventaccumulation of the Bt protein in seed. Hence the protein encoded by theintroduced gene would be present in all tissues except the kernel.

Expression of some genes in transgenic plants will be desired only underspecified conditions. For example, it is proposed that expression ofcertain genes that confer resistance environmental stress factors suchas drought will be desired only under actual stress conditions. It iscontemplated that expression of such genes throughout a plantsdevelopment may have detrimental effects. It is known that a largenumber of genes exist that respond to the environment. For example,expression of some genes such as rbcS, encoding the small subunit ofribulose bisphosphate carboxylase, is regulated by light as mediatedthrough phytochrome. Other genes are induced by secondary stimuli. Forexample, synthesis of abscisic acid (ABA) is induced by certainenvironmental factors, including but not limited to water stress. Anumber of genes have been shown to be induced by ABA (Skriver and Mundy,1990). It is also anticipated that expression of genes conferringresistance to insect predation would be desired only under conditions ofactual insect infestation. Therefore, for some desired traits inducibleexpression of genes in transgenic plants will be desired.

Expression of a gene in a transgenic plant will be desired only in acertain time period during the development of the plant. Developmentaltiming is frequently correlated with tissue specific gene expression.For example, expression of zein storage proteins is initiated in theendosperm about 15 days after pollination.

Additionally, vectors may be constructed and employed in theintracellular targeting of a specific gene product within the cells of atransgenic plant or in directing a protein to the extracellularenvironment. This will generally be achieved by joining a DNA sequenceencoding a transit or signal peptide sequence to the coding sequence ofa particular gene. The resultant transit, or signal, peptide willtransport the protein to a particular intracellular, or extracellulardestination, respectively, and will then be post-translationallyremoved. Transit or signal peptides act by facilitating the transport ofproteins through intracellular membranes, e.g., vacuole, vesicle,plastid and mitochondrial membranes, whereas signal peptides directproteins through the extracellular membrane.

A particular example of such a use concerns the direction of a herbicideresistance gene, such as the EPSPS gene, to a particular organelle suchas the chloroplast rather than to the cytoplasm. This is exemplified bythe use of the rbcs transit peptide which confers plastid-specifictargeting of proteins. In addition, it is proposed that it may bedesirable to target certain genes responsible for male sterility to themitochondria, or to target certain genes for resistance tophytopathogenic organisms to the extracellular spaces, or to targetproteins to the vacuole.

By facilitating the transport of the protein into compartments insideand outside the cell, these sequences may increase the accumulation ofgene product protecting them from proteolytic degradation. Thesesequences also allow for additional mRNA sequences from highly expressedgenes to be attached to the coding sequence of the genes. Since mRNAbeing translated by ribosomes is more stable than naked mRNA, thepresence of translatable mRNA in front of the gene may increase theoverall stability of the mRNA transcript from the gene and therebyincrease synthesis of the gene product. Since transit and signalsequences are usually post-translationally removed from the initialtranslation product, the use of these sequences allows for the additionof extra translated sequences that may not appear on the finalpolypeptide. Targeting of certain proteins may be desirable in order toenhance the stability of the protein (U.S. Pat. No. 5,545,818).

It may be useful to target DNA itself within a cell. For example, it maybe useful to target introduced DNA to the nucleus as this may increasethe frequency of transformation. Within the nucleus itself it would beuseful to target a gene in order to achieve site specific integration.For example, it would be useful to have an gene introduced throughtransformation replace an existing gene in the cell.

Other elements include those that can be regulated by endogenous orexogenous agents, e.g., by zinc finger proteins, including naturallyoccurring zinc finger proteins or chimeric zinc finger proteins (see,e.g., U.S. Pat. No. 5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO98/53057; WO 98/53058; WO 00/23464; WO 95/19431; and WO 98/54311) ormyb-like transcription factors. For example, a chimeric zinc fingerprotein may include amino acid sequences which bind to a specific DNAsequence (the zinc finger) and amino acid sequences that activate (e.g.,GAL 4 sequences) or repress the transcription of the sequences linked tothe specific DNA sequence.

3. Methods for Mutagenizing DNA

It is specifically contemplated by the inventors that one couldmutagenize DNA having a promoter or open reading frame to, for example,potentially improve the utility of the DNA for expression of transgenesin plants. The mutagenesis can be carried out at random and themutagenized sequences screened for activity in a trial-by-errorprocedure. Alternatively, particular sequences which provide thepromoter with desirable expression characteristics, or a promoter withexpression enhancement activity, could be identified and these orsimilar sequences introduced into the sequences via mutation. It isfurther contemplated that one could mutagenize these sequences in orderto enhance their expression of transgenes in a particular species.

The means for mutagenizing a DNA segment of the current invention arewell-known to those of skill in the art. As indicated, modifications maybe made by random or site-specific mutagenesis procedures. The DNA maybe modified by altering its structure through the addition or deletionof one or more nucleotides from the sequence which encodes thecorresponding unmodified sequences.

Mutagenesis may be performed in accordance with any of the techniquesknown in the art, such as, and not limited to, synthesizing anoligonucleotide having one or more mutations within the sequence of aparticular regulatory region. In particular, site-specific mutagenesisis a technique useful in the preparation of promoter mutants, throughspecific mutagenesis of the underlying DNA. The technique furtherprovides a ready ability to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 toabout 75 nucleotides or more in length is preferred, with about 10 toabout 25 or more residues on both sides of the junction of the sequencebeing altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by various publications. As will be appreciated,the technique typically employs a phage vector which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage are readily commercially available and their use is generally wellknown to those skilled in the art.

Double stranded plasmids also are routinely employed in site directedmutagenesis which eliminates the step of transferring the gene ofinterest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the promoter. An oligonucleotideprimer bearing the desired mutated sequence is prepared, generallysynthetically. This primer is then annealed with the single-strandedvector, and subjected to DNA polymerizing enzymes such as E. colipolymerase I Klenow fragment, in order to complete the synthesis of themutation-bearing strand. Thus, a heteroduplex is formed wherein onestrand encodes the original non-mutated sequence and the second strandbears the desired mutation.

This heteroduplex vector is then used to transform or transfectappropriate cells, such as E. coli cells, and cells are selected whichinclude recombinant vectors bearing the mutated sequence arrangement.Vector DNA can then be isolated from these cells and used for planttransformation. A genetic selection scheme was devised by Kunkel et al.(1987) to enrich for clones incorporating mutagenic oligonucleotides.Alternatively, the use of PCR with commercially available thermostableenzymes such as Taq polymerase may be used to incorporate a mutagenicoligonucleotide primer into an amplified DNA fragment that can then becloned into an appropriate cloning or expression vector. ThePCR-mediated mutagenesis procedures of Tomic et al. (1990) and Upenderet al. (1995) provide two examples of such protocols. A PCR employing athermostable ligase in addition to a thermostable polymerase also may beused to incorporate a phosphorylated mutagenic oligonucleotide into anamplified DNA fragment that may then be cloned into an appropriatecloning or expression vector. The mutagenesis procedure described byMichael (1994) provides an example of one such protocol.

The preparation of sequence variants of DNA segments using site-directedmutagenesis is provided as a means of producing potentially usefulspecies and is not meant to be limiting as there are other ways in whichsequence variants of DNA sequences may be obtained. For example,recombinant vectors encoding the desired promoter sequence may betreated with mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

In addition, an unmodified or modified nucleotide sequence of thepresent invention can be varied by shuffling the sequence of theinvention. To test for a function of variant DNA sequences according tothe invention, the sequence of interest is operably linked to aselectable or screenable marker gene and expression of the marker geneis tested in transient expression assays with protoplasts or in stablytransformed plants. It is known to the skilled artisan that DNAsequences capable of driving expression of an associated nucleotidesequence are build in a modular way. Accordingly, expression levels fromshorter DNA fragments may be different than the one from the longestfragment and may be different from each other. For example, deletion ofa down-regulating upstream element will lead to an increase in theexpression levels of the associated nucleotide sequence while deletionof an up-regulating element will decrease the expression levels of theassociated nucleotide sequence. It is also known to the skilled artisanthat deletion of development-specific or a tissue-specific element willlead to a temporally or spatially altered expression profile of theassociated nucleotide sequence.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” also is intended to refer to a process thatinvolves the template-dependent extension of a primer molecule. The termtemplate-dependent process refers to nucleic acid synthesis of an RNA ora DNA molecule wherein the sequence of the newly synthesized strand ofnucleic acid is dictated by the well-known rules of complementary basepairing (see, for example, Watson and Rarnstad, 1987). Typically, vectormediated methodologies involve the introduction of the nucleic acidfragment into a DNA or RNA vector, the clonal amplification of thevector, and the recovery of the amplified nucleic acid fragment.Examples of such methodologies are provided by U.S. Pat. No. 4,237,224.A number of template dependent processes are available to amplify thetarget sequences of interest present in a sample, such methods beingwell known in the art and specifically disclosed herein below.

Where a clone comprising a promoter has been isolated in accordance withthe instant invention, one may wish to delimit the essential promoterregions within the clone. One efficient, targeted means for preparingmutagenizing promoters relies upon the identification of putativeregulatory elements within the promoter sequence. This can be initiatedby comparison with promoter sequences known to be expressed in similartissue-specific or developmentally unique manner. Sequences which areshared among promoters with similar expression patterns are likelycandidates for the binding of transcription factors and are thus likelyelements which confer expression patterns. Confirmation of theseputative regulatory elements can be achieved by deletion analysis ofeach putative regulatory region followed by functional analysis of eachdeletion construct by assay of a reporter gene which is functionallyattached to each construct. As such, once a starting promoter sequenceis provided, any of a number of different deletion mutants of thestarting promoter could be readily prepared.

As indicated above, deletion mutants, deletion mutants of the promoterof the invention also could be randomly prepared and then assayed. Withthis strategy, a series of constructs are prepared, each containing adifferent portion of the clone (a subclone), and these constructs arethen screened for activity. A suitable means for screening for activityis to attach a deleted promoter or intron construct which contains adeleted segment to a selectable or screenable marker, and to isolateonly those cells expressing the marker gene. In this way, a number ofdifferent, deleted promoter constructs are identified which still retainthe desired, or even enhanced, activity. The smallest segment which isrequired for activity is thereby identified through comparison of theselected constructs. This segment may then be used for the constructionof vectors for the expression of exogenous genes.

B. Marker Genes

In order to improve the ability to identify transformants, one maydesire to employ a selectable or screenable marker gene as, or inaddition to, the expressible gene of interest. “Marker genes” are genesthat impart a distinct phenotype to cells expressing the marker gene andthus allow such transformed cells to be distinguished from cells that donot have the marker. Such genes may encode either a selectable orscreenable marker, depending on whether the marker confers a trait whichone can ‘select’ for by chemical means, i.e., through the use of aselective agent (e.g., a herbicide, antibiotic, or the like), or whetherit is simply a trait that one can identify through observation ortesting, i.e., by ‘screening’ (e.g., the R-locus trait, the greenfluorescent protein (GFP)). Of course, many examples of suitable markergenes are known to the art and can be employed in the practice of theinvention.

Included within the terms selectable or screenable marker genes are alsogenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers which encode a secretable antigen that can be identifiedby antibody interaction, or even secretable enzymes which can bedetected by their catalytic activity. Secretable proteins fall into anumber of classes, including small, diffusible proteins detectable,e.g., by ELISA; small active enzymes detectable in extracellularsolution (e.g., alpha-amylase, beta-lactamase, phosphinothricinacetyltransferase); and proteins that are inserted or trapped in thecell wall (e.g., proteins that include a leader sequence such as thatfound in the expression unit of extensin or tobacco PRS).

With regard to selectable secretable markers, the use of a gene thatencodes a protein that becomes sequestered in the cell wall, and whichprotein includes a unique epitope is considered to be particularlyadvantageous. Such a secreted antigen marker would ideally employ anepitope sequence that would provide low background in plant tissue, apromoter-leader sequence that would impart efficient expression andtargeting across the plasma membrane, and would produce protein that isbound in the cell wall and yet accessible to antibodies. A normallysecreted wall protein modified to include a unique epitope would satisfyall such requirements.

One example of a protein suitable for modification in this manner isextensin, or hydroxyproline rich glycoprotein (HPRG). For example, themaize HPRG (Steifel et al., 1990) molecule is well characterized interms of molecular biology, expression and protein to structure.However, any one of a variety of ultilane and/or glycine-rich wallproteins (Keller et al., 1989) could be modified by the addition of anantigenic site to create a screenable marker.

One exemplary embodiment of a secretable screenable marker concerns theuse of a maize sequence encoding the wall protein HPRG, modified toinclude a 15 residue epitope from the pro-region of murine interleukin,however, virtually any detectable epitope may be employed in suchembodiments, as selected from the extremely wide variety ofantigen-antibody combinations known to those of skill in the art. Theunique extracellular epitope can then be straightforwardly detectedusing antibody labeling in conjunction with chromogenic or fluorescentadjuncts.

Elements of the present disclosure may be exemplified in detail throughthe use of the bar and/or GUS genes, and also through the use of variousother markers. Of course, in light of this disclosure, numerous otherpossible selectable and/or screenable marker genes will be apparent tothose of skill in the art in addition to the one set forth hereinbelow.Therefore, it will be understood that the following discussion isexemplary rather than exhaustive. In light of the techniques disclosedherein and the general recombinant techniques which are known in theart, the present invention renders possible the introduction of anygene, including marker genes, into a recipient cell to generate atransformed plant.

1. Selectable Markers

Possible selectable markers for use in connection with the presentinvention include, but are not limited to, a neo gene which codes forkanamycin resistance and can be selected for using kanamycin, G418,paromomycin, and the like; a bar gene which codes for bialaphos orphosphinothricin resistance; a gene which encodes an altered EPSPsynthase protein (Hinchee et al., 1988) thus conferring glyphosateresistance; a nitrilase gene such as bxn from Klebsiella ozaenae whichconfers resistance to bromoxynil (Stalker et al., 1988); a mutantacetolactate synthase gene (ALS) which confers resistance toimidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EuropeanPatent Application 154,204, 1985); a methotrexate-resistant DHFR gene(Thillet et al., 1988); a dalapon dehalogenase gene that confersresistance to the herbicide dalapon; a mutated anthranilate synthasegene that confers resistance to 5-methyl tryptophan. Preferredselectable marker genes encode phosphinothricin acetyltransferase;glyphosate resistant EPSPS, aminoglycoside phosphotransferase;hygromycin phosphotransferase, or neomycin phosphotransferase. Where amutant EPSP synthase gene is employed, additional benefit may berealized through the incorporation of a suitable chloroplast transitpeptide, CTP (European Patent Application 0,218,571, 1987).

An illustrative embodiment of a selectable marker gene capable of beingused in systems to select transformants is the genes that encode theenzyme phosphinothricin acetyltransferase, such as the bar gene fromStreptomyces hygroscopicus or the pat gene from Streptomycesviridochromogenes. The enzyme phosphinothricin acetyl transferase (PAT)inactivates the active ingredient in the herbicide bialaphos,phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami etal., 1986; Twell et al., 1989) causing rapid accumulation of ammonia andcell death. The success in using this selective system in conjunctionwith monocots was particularly surprising because of the majordifficulties which have been reported in transformation of cereals.

Where one desires to employ a bialaphos resistance gene in the practiceof the invention, a particularly useful gene for this purpose is the baror pat genes obtainable from species of Streptomyces (e.g., ATCC No.21,705). The cloning of the bar gene has been described (Murakami etal., 1986; Thompson et al., 1987) as has the use of the bar gene in thecontext of plants other than monocots (De Block et al., 1987; De Blocket al., 1989).

Selection markers resulting in positive selection, such as aphosphomannose isomerase gene, as described in patent application WO93/05163, may also be used. Alternative genes to be used for positiveselection are described in WO 94/20627 and encode xyloisomerases andphosphomanno-isomerases such as mannose-6-phosphate isomerase andmannose-1′-phosphate isomerase; phosphomanno mutase; mannose epimerasessuch as those which convert carbohydrates to mannose or mannose tocarbohydrates such as glucose or galactose; phosphatases such as mannose or xylose phosphatase, mannose-6-phosphatase andmannose-1-phosphatase, and permeases which are involved in the transportof mannose, or a derivative, or a precursor thereof into the cell.Transformed cells are identified without damaging or killing thenon-transformed cells in the population and without co-introduction ofantibiotic or herbicide resistance genes. As described in WO 93/05163,in addition to the fact that the need for antibiotic or herbicideresistance genes is eliminated, it has been shown that the positiveselection method is often far more efficient than traditional negativeselection.

2. Screenable Markers

Screenable markers that may be employed include, but are not limited to,a beta-glucuronidase (GUS) or uidA gene which encodes an enzyme forwhich various chromogenic substrates are known; an R-locus gene, whichencodes a product that regulates the production of anthocyanin pigments(red color) in plant tissues (Dellaporta et al., 1988); a beta-lactamasegene (Sutcliffe, 1978), which encodes an enzyme for which variouschromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a xyle gene (Zukowsky et al., 1983) which encodes acatechol dioxygenase that can convert chromogenic catechols; anα-amylase gene (Ikuta et al., 1990); a tyrosinase gene (Katz et al.,1983) which encodes an enzyme capable of oxidizing tyrosine to DOPA anddopaquinone which in turn condenses to form the easily detectablecompound melanin; a β-galactosidase gene, which encodes an enzyme forwhich there are chromogenic substrates; a luciferase (lux) gene (Ow etal., 1986), which allows for bioluminescence detection; or even anaequorin gene (Prasher et al., 1985), which may be employed incalcium-sensitive bioluminescence detection, or a green fluorescentprotein gene (Niedz et al., 1995).

Genes from the maize R gene complex are contemplated to be particularlyuseful as screenable markers. The R gene complex in maize encodes aprotein that acts to regulate the production of anthocyanin pigments inmost seed and plant tissue. A gene from the R gene complex was appliedto maize transformation, because the expression of this gene intransformed cells does not harm the cells. Thus, an R gene introducedinto such cells will cause the expression of a red pigment and, ifstably incorporated, can be visually scored as a red sector. If a maizeline is carries dominant ultila for genes encoding the enzymaticintermediates in the anthocyanin biosynthetic pathway (C2, A1, A2, Bz1and Bz2) (Roth et al., 1990), but carries a recessive allele at the Rlocus, transformation of any cell from that line with R will result inred pigment formation. Exemplary lines include Wisconsin 22 whichcontains the rg-Stadler allele and TR112, a K55 derivative which is r-g,b, P1. Alternatively any genotype of maize can be utilized if the C1 andR alleles are introduced together.

It is further proposed that R gene regulatory regions may be employed inchimeric constructs in order to provide mechanisms for controlling theexpression of chimeric genes. More diversity of phenotypic expression isknown at the R locus than at any other locus (Coe et al., 1988). It iscontemplated that regulatory regions obtained from regions 5′ to thestructural R gene would be valuable in directing the expression ofgenes, e.g., insect resistance, drought resistance, herbicide toleranceor other protein coding regions. For the purposes of the presentinvention, it is believed that any of the various R gene family membersmay be successfully employed (e.g., P, S, Lc, etc.). However, the mostpreferred will generally be Sn (particularly Sn:bo13). Sn is a dominantmember of the R gene complex and is functionally similar to the R and Bloci in that Sn controls the tissue specific deposition of anthocyaninpigments in certain seedling and plant cells, therefore, its phenotypeis similar to R.

A further screenable marker contemplated for use in the presentinvention is firefly luciferase, encoded by the lux gene. The presenceof the lux gene in transformed cells may be detected using, for example,X-ray film, scintillation counting, fluorescent spectrophotometry,low-light video cameras, photon counting cameras or multiwellluminometry. It is also envisioned that this system may be developed forpopulational screening for bioluminescence, such as on tissue cultureplates, or even for whole plant screening. Where use of a screenablemarker gene such as lux or GFP is desired, benefit may be realized bycreating a gene fusion between the screenable marker gene and aselectable marker gene, for example, a GFP-NPTII gene fusion. This couldallow, for example, selection of transformed cells followed by screeningof transgenic plants or seeds.

C. Exogenous Genes for Modification of Plant Phenotypes

Genes of interest are reflective of the commercial markets and interestsof those involved in the development of the crop. Crops and markets ofinterest changes, and as developing nations open up world markets, newcrops and technologies will also emerge. In addition, as theunderstanding of agronomic traits and characteristics such as yield andheterosis increase, the choice of genes for transformation will changeaccordingly. General categories of genes of interest include, forexample, those genes involved in information, such as zinc fingers,those involved in communication, such as kinases, and those involved inhousekeeping, such as heat shock proteins. More specific categories oftransgenes, for example, include genes encoding important traits foragronomics, insect resistance, disease resistance, herbicide resistance,sterility, grain characteristics, and commercial products. Genes ofinterest include, generally, those involved in starch, oil,carbohydrate, or nutrient metabolism, as well as those affecting kernelsize, sucrose loading, zinc finger proteins, see, e.g., U.S. Pat. No.5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO 98/53057; WO98/53058; WO 00/23464; WO 95/19431; and WO 98/54311, and the like.

One skilled in the art recognizes that the expression level andregulation of a transgene in a plant can vary significantly from line toline. Thus, one has to test several lines to find one with the desiredexpression level and regulation. Once a line is identified with thedesired regulation specificity of a chimeric Cre transgene, it can becrossed with lines carrying different inactive replicons or inactivetransgene for activation.

Other sequences which may be linked to the gene of interest whichencodes a polypeptide are those which can target to a specificorganelle, e.g., to the mitochondria, nucleus, or plastid, within theplant cell. Targeting can be achieved by providing the polypeptide withan appropriate targeting peptide sequence, such as a secretory signalpeptide (for secretion or cell wall or membrane targeting, a plastidtransit peptide, a chloroplast transit peptide, e.g., the chlorophylla/b binding protein, a mitochondrial target peptide, a vacuole targetingpeptide, or a nuclear targeting peptide, and the like. For example, thesmall subunit of ribulose bisphosphate carboxylase transit peptide, theEPSPS transit peptide or the dihydrodipicolinic acid synthase transitpeptide may be used. For examples of plastid organelle targetingsequences (see WO 00/12732). Plastids are a class of plant organellesderived from proplastids and include chloroplasts, leucoplasts,amyloplasts, and chromoplasts. The plastids are major sites ofbiosynthesis in plants. In addition to photosynthesis in thechloroplast, plastids are also sites of lipid biosynthesis, nitratereduction to ammonium, and starch storage. And while plastids containtheir own circular genome, most of the proteins localized to theplastids are encoded by the nuclear genome and are imported into theorganelle from the cytoplasm.

Transgenes used with the present invention will often be genes thatdirect the expression of a particular protein or polypeptide product,but they may also be non-expressible DNA segments, e.g., transposonssuch as Ds that do no direct their own transposition. As used herein, an“expressible gene” is any gene that is capable of being transcribed intoRNA (e.g., mRNA, antisense RNA, etc.) or translated into a protein,expressed as a trait of interest, or the like, etc., and is not limitedto selectable, screenable or non-selectable marker genes. The inventionalso contemplates that, where both an expressible gene that is notnecessarily a marker gene is employed in combination with a marker gene,one may employ the separate genes on either the same or different DNAsegments for transformation. In the latter case, the different vectorsare delivered concurrently to recipient cells to maximizecotransformation.

The choice of the particular DNA segments to be delivered to therecipient cells will often depend on the purpose of the transformation.One of the major purposes of transformation of crop plants is to addsome commercially desirable, agronomically important traits to theplant. Such traits include, but are not limited to, herbicide resistanceor tolerance; insect resistance or tolerance; disease resistance ortolerance (viral, bacterial, fungal, nematode); stress tolerance and/orresistance, as exemplified by resistance or tolerance to drought, heat,chilling, freezing, excessive moisture, salt stress; oxidative stress;increased yields; food content and makeup; physical appearance; malesterility; drydown; standability; prolificacy; starch properties; oilquantity and quality; and the like. One may desire to incorporate one ormore genes conferring any such desirable trait or traits, such as, forexample, a gene or genes encoding pathogen resistance.

In certain embodiments, the present invention contemplates thetransformation of a recipient cell with more than one advantageoustransgene. Two or more transgenes can be supplied in a singletransformation event using either distinct transgene-encoding vectors,or using a single vector incorporating two or more gene codingsequences. For example, plasmids bearing the bar and aroA expressionunits in either convergent, divergent, or colinear orientation, areconsidered to be particularly useful. Further preferred combinations arethose of an insect resistance gene, such as a Bt gene, along with aprotease inhibitor gene such as pinII, or the use of bar in combinationwith either of the above genes. Of course, any two or more transgenes ofany description, such as those conferring herbicide, insect, disease(viral, bacterial, fungal, nematode) or drought resistance, malesterility, drydown, standability, prolificacy, starch properties, oilquantity and quality, or those increasing yield or nutritional qualitymay be employed as desired.

1. Herbicide Resistance

The genes encoding phosphinothricin acetyltransferase (bar and pat),glyphosate tolerant EPSP synthase genes, the glyphosate degradativeenzyme gene gox encoding glyphosate oxidoreductase, deh (encoding adehalogenase enzyme that inactivates dalapon), herbicide resistant(e.g., sulfonylurea and imidazolinone) acetolactate synthase, and bxngenes (encoding a nitrilase enzyme that degrades bromoxynil) are goodexamples of herbicide resistant genes for use in transformation. The barand pat genes code for an enzyme, phosphinothricin acetyltransferase(PAT), which inactivates the herbicide phosphinothricin and preventsthis compound from inhibiting glutamine synthetase enzymes. The enzyme5-enolpyruvyishikimate 3-phosphate synthase (EPSP Synthase), is normallyinhibited by the herbicide N-(phosphonomethyl)glycine (glyphosate).However, genes are known that encode glyphosate-resistant EPSP Synthaseenzymes.

These genes are particularly contemplated for use in monocottransformation. The deh gene encodes the enzyme dalapon dehalogenase andconfers resistance to the herbicide dalapon. The bxn gene codes for aspecific nitrilase enzyme that converts bromoxynil to a non-herbicidaldegradation product.

2. Insect Resistance

An important aspect of the present invention concerns the introductionof insect resistance-conferring genes into plants. Potential insectresistance genes which can be introduced include Bacillus thuringiensiscrystal toxin genes or Bt genes (Watrud et al., 1985). Bt genes mayprovide resistance to lepidopteran or coleopteran pests such as EuropeanCorn Borer (ECB) and corn rootworm (CRW). Preferred Bt toxin genes foruse in such embodiments include the CryIA(b) and CryIA(c) genes.Endotoxin genes from other species of B. thuringiensis which affectinsect growth or development may also be employed in this regard.

The poor expression of Bt toxin genes in plants is a well-documentedphenomenon, and the use of different promoters, fusion proteins, andleader sequences has not led to significant increases in Bt proteinexpression (Vaeck et al., 1989; Barton et al., 1987). It is thereforecontemplated that the most advantageous Bt genes for use in thetransformation protocols disclosed herein will be those in which thecoding sequence has been modified to effect increased expression inplants, and more particularly, those in which maize preferred codonshave been used. Examples of such modified Bt toxin genes include thevariant Bt CryIA(b) gene termed lab6 (Perlak et al., 1991) and thesynthetic CryIA(c) genes termed 1800a and 1800b.

Protease inhibitors may also provide insect resistance (Johnson et al.,1989), and will thus have utility in plant transformation. The use of aprotease inhibitor II gene, pinII, from tomato or potato is envisionedto be particularly useful. Even more advantageous is the use of a pinIIgene in combination with a Bt toxin gene, the combined effect of whichhas been discovered by the present inventors to produce synergisticinsecticidal activity. Other genes which encode inhibitors of theinsects' digestive system, or those that encode enzymes or co-factorsthat facilitate the production of inhibitors, may also be useful. Thisgroup may be exemplified by oryzacystatin and amylase inhibitors, suchas those from wheat and barley.

Also, genes encoding lectins may confer additional or alternativeinsecticide propenies. Lectins (originally termed phytohemagglutinins)are multivalent carbohydrate-binding proteins which have the ability toagglutinate red blood cells from a range of species. Lectins have beenidentified recently as insecticidal agents with activity againstweevils, ECB and rootworm (Murdock et al., 1990; Czapla and Lang, 1990).Lectin genes contemplated to be useful include, for example, barley andwheat germ agglutinin (WGA) and rice lectins (Gatehouse et al., 1984),with WGA being preferred.

Genes controlling the production of large or small polypeptides activeagainst insects when introduced into the insect pests, such as, e.g.,lytic peptides, peptide hormones and toxins and venoms, form anotheraspect of the invention. For example, it is contemplated that theexpression of juvenile hormone esterase, directed towards specificinsect pests, may also result in insecticidal activity, or perhaps causecessation of metamorphosis (Hammock et al., 1990).

Transgenic plants expressing genes which encode enzymes that affect theintegrity of the insect cuticle form yet another aspect of theinvention. Such genes include those encoding, e.g., chitinase,proteases, lipases and also genes for the production of nikkomycin, acompound that inhibits chitin synthesis, the introduction of any ofwhich is contemplated to produce insect resistant maize plants. Genesthat code for activities that affect insect molting, such thoseaffecting the production of ecdysteroid UDP-glucosyl transferase, alsofall within the scope of the useful transgenes of the present invention.

Genes that code for enzymes that facilitate the production of compoundsthat reduce the nutritional quality of the host plant to insect pestsare also encompassed by the present invention. It may be possible, forinstance, to confer insecticidal activity on a plant by altering itssterol composition. Sterols are obtained by insects from their diet andare used for hormone synthesis and membrane stability. Thereforealterations in plant sterol composition by expression of novel genes,e.g., those that directly promote the production of undesirable sterolsor those that convert desirable sterols into undesirable forms, couldhave a negative effect on insect growth and/or development and henceendow the plant with insecticidal activity. Lipoxygenases are naturallyoccurring plant enzymes that have been shown to exhibit anti-nutritionaleffects on insects and to reduce the nutritional quality of their diet.Therefore, further embodiments of the invention concern transgenicplants with enhanced lipoxygenase activity which may be resistant toinsect feeding.

The present invention also provides methods and compositions by which toachieve qualitative or quantitative changes in plant secondarymetabolites. One example concerns transforming plants to produce DIMBOAwhich, it is contemplated, will confer resistance to European cornborer, rootworm and several other maize insect pests. Candidate genesthat are particularly considered for use in this regard include thosegenes at the bx locus known to be involved in the synthetic DIMBOApathway (Dunn et al., 1981). The introduction of genes that can regulatethe production of maysin, and genes involved in the production ofdhurrin in sorghum, is also contemplated to be of use in facilitatingresistance to earworm and rootworm, respectively.

Tripsacum dactyloides is a species of grass that is resistant to certaininsects, including corn root worm. It is anticipated that genes encodingproteins that are toxic to insects or are involved in the biosynthesisof compounds toxic to insects will be isolated from Tripsacum and thatthese novel genes will be useful in conferring resistance to insects. Itis known that the basis of insect resistance in Tripsacum is genetic,because said resistance has been transferred to Zea mays via sexualcrosses (Branson and Guss, 1972).

Further genes encoding proteins characterized as having potentialinsecticidal activity may also be used as transgenes in accordanceherewith. Such genes include, for example, the cowpea trypsin inhibitor(CPTI; Hilder et al., 1987) which may be used as a rootworm deterrent;genes encoding avermectin (Campbell, 1989; Ikeda et al., 1987) which mayprove particularly useful as a corn rootworm deterrent; ribosomeinactivating protein genes; and even genes that regulate plantstructures. Transgenic maize including anti-insect antibody genes andgenes that code for enzymes that can covert a non-toxic insecticide(pro-insecticide) applied to the outside of the plant into aninsecticide inside the plant are also contemplated.

3. Environment or Stress Resistance

Improvement of a plant's ability to tolerate various environmentalstresses such as, but not limited to, drought, excess moisture,chilling, freezing, high temperature, salt, and oxidative stress, canalso be effected through expression of heterologous, or overexpressionof homologous genes. Benefits may be realized in terms of increasedresistance to freezing temperatures through the introduction of an“antifreeze” protein such as that of the Winter Flounder (Cutler et al.,1989) or synthetic gene derivatives thereof. Improved chilling tolerancemay also be conferred through increased expression ofglycerol-3-phosphate acetyltransferase in chloroplasts (Murata et al.,1992; Wolter et al., 1992). Resistance to oxidative stress (oftenexacerbated by conditions such as chilling temperatures in combinationwith high light intensities) can be conferred by expression ofsuperoxide dismutase (Gupta et al., 1993), and may be improved byglutathione reductase (Bowler et al., 1992). Such strategies may allowfor tolerance to freezing in newly emerged fields as well as extendinglater maturity higher yielding varieties to earlier relative maturityzones.

Expression of novel genes that favorably effect plant water content,total water potential, osmotic potential, and turgor can enhance theability of the plant to tolerate drought. As used herein, the terms“drought resistance” and “drought tolerance” are used to refer to aplants increased resistance or tolerance to stress induced by areduction in water availability, as compared to normal circumstances,and the ability of the plant to function and survive in lower-waterenvironments, and perform in a relatively superior manner. In thisaspect of the invention it is proposed, for example, that the expressionof a gene encoding the biosynthesis of osmotically-active solutes canimpart protection against drought. Within this class of genes are DNAsencoding mannitol dehydrogenase (Lee and Saier, 1982) andtrehalose-6-phosphate synthase (Kaasen et al., 1992). Through thesubsequent action of native phosphatases in the cell or by theintroduction and coexpression of a specific phosphatase, theseintroduced genes will result in the accumulation of either mannitol ortrehalose, respectively, both of which have been well documented asprotective compounds able to mitigate the effects of stress. Mannitolaccumulation in transgenic tobacco has been verified and preliminaryresults indicate that plants expressing high levels of this metaboliteare able to tolerate an applied osmotic stress (Tarczynski et al., citedsupra (1992), 1993).

Similarly, the efficacy of other metabolites in protecting either enzymefunction (e.g. alanopine or propionic acid) or membrane integrity (e.g.,alanopine) has been documented (Loomis et al., 1989), and thereforeexpression of gene encoding the biosynthesis of these compounds canconfer drought resistance in a manner similar to or complimentary tomannitol. Other examples of naturally occurring metabolites that areosmotically active and/or provide some direct protective effect duringdrought and/or desiccation include sugars and sugar derivatives such asfructose, erythritol (Coxson et al., 1992), sorbitol, dulcitol (Karstenet al., 1992), glucosylglycerol (Reed et al., 1984; Erdmann et al.,1992), sucrose, stachyose (Koster and Leopold, 1988; Blackman et al.,1992), ononitol and pinitol (Vernon and Bohnert, 1992), and raffinose(Bernal-Lugo and Leopold, 1992). Other osmotically active solutes whichare not sugars include, but are not limited to, proline andglycine-betaine (Wyn-Jones and Storey, 1981). Continued canopy growthand increased reproductive fitness during times of stress can beaugmented by introduction and expression of genes such as thosecontrolling the osmotically active compounds discussed above and othersuch compounds, as represented in one exemplary embodiment by the enzymemyoinositol O-methyltransferase.

It is contemplated that the expression of specific proteins may alsoincrease drought tolerance. Three classes of Late Embryogenic Proteinshave been assigned based on structural similarities (see Dure et al.,1989). All three classes of these proteins have been demonstrated inmaturing (i.e., desiccating) seeds. Within these 3 types of proteins,the Type-II (dehydrin-type) have generally been implicated in droughtand/or desiccation tolerance in vegetative plant parts (i.e. Mundy andChua, 1988; Piatkowski et al., 1990; Yamaguchi-Shinozaki et al., 1992).Recently, expression of a Type-III LEA (HVA-1) in tobacco was found toinfluence plant height, maturity and drought tolerance (Fitzpatrick,1993). Expression of structural genes from all three groups maytherefore confer drought tolerance. Other types of proteins inducedduring water stress include thiol proteases, aldolases and transmembranetransporters (Guerrero et al., 1990), which may confer variousprotective and/or repair-type functions during drought stress. Theexpression of a gene that effects lipid biosynthesis and hence membranecomposition can also be useful in conferring drought resistance on theplant.

Many genes that improve drought resistance have complementary modes ofaction. Thus, combinations of these genes might have additive and/orsynergistic effects in improving drought resistance in plants. Many ofthese genes also improve freezing tolerance (or resistance); thephysical stresses incurred during freezing and drought are similar innature and may be mitigated in similar fashion. Benefit may be conferredvia constitutive expression of these genes, but the preferred means ofexpressing these novel genes may be through the use of a turgor-inducedpromoter (such as the promoters for the turgor-induced genes describedin Guerrero et al. 1990 and Shagan et al., 1993). Spatial and temporalexpression patterns of these genes may enable maize to better withstandstress.

Expression of genes that are involved with specific morphological traitsthat allow for increased waler extractions from drying soil would be ofbenefit. For example, introduction and expression of genes that alterroot characteristics may enhance water uptake. Expression of genes thatenhance reproductive fitness during times of stress would be ofsignificant value. For example, expression of DNAs that improve thesynchrony of pollen shed and receptiveness of the female flower parts,i.e., silks, would be of benefit. In addition, expression of genes thatminimize kernel abortion during times of stress would increase theamount of grain to be harvested and hence be of value. Regulation ofcytokinin levels in monocots, such as maize, by introduction andexpression of an isopentenyl transferase gene with appropriateregulatory sequences can improve monocot stress resistance and yield(Gan et al., Science, 270:1986 (1995)).

Given the overall role of water in determining yield, it is contemplatedthat enabling plants to utilize water more efficiently, through theintroduction and expression of novel genes, will improve overallperformance even when soil water availability is not limiting. Byintroducing genes that improve the ability of plants to maximize waterusage across a full range of stresses relating to water availability,yield stability or consistency of yield performance may be realized.

4. Disease Resistance

It is proposed that increased resistance to diseases may be realizedthrough introduction of genes into plants period. It is possible toproduce resistance to diseases caused by viruses, bacteria, fungi, rootpathogens, insects and nematodes. It is also contemplated that controlof mycotoxin producing organisms may be realized through expression ofintroduced genes.

Resistance to viruses may be produced through expression of novel genes.For example, it has been demonstrated that expression of a viral coatprotein in a transgenic plant can impart resistance to infection of theplant by that virus and perhaps other closely related viruses (Cuozzo etal., 1988, Hemenway et al., 1988, Abel et al., 1986). It is contemplatedthat expression of antisense genes targeted at essential viral functionsmay impart resistance to said virus. For example, an antisense genetargeted at the gene responsible for replication of viral nucleic acidmay inhibit said replication and lead to resistance to the virus. It isbelieved that interference with other viral functions through the use ofantisense genes may also increase resistance to viruses. Further it isproposed that it may be possible to achieve resistance to virusesthrough other approaches, including, but not limited to the use ofsatellite viruses.

It is proposed that increased resistance to diseases caused by bacteriaand fungi may be realized through introduction of novel genes. It iscontemplated that genes encoding so-called “peptide antibiotics,”pathogenesis related (PR) proteins, toxin resistance, and proteinsaffecting host-pathogen interactions such as morphologicalcharacteristics will be useful. Peptide antibiotics are polypeptidesequences which are inhibitory to growth of bacteria and othermicroorganisms. For example, the classes of peptides referred to ascecropins and magainins inhibit growth of many species of bacteria andfungi. It is proposed that expression of PR proteins in plants may beuseful in conferring resistance to bacterial disease. These genes areinduced following pathogen attack on a host plant and have been dividedinto at least five classes of proteins (Bol et al., 1990). Includedamongst the PR proteins are beta1,3-glucanases, chitinases, and osmotinand other proteins that are believed to function in plant resistance todisease organisms. Other genes have been identified that have antifungalproperties, e.g., UDA (stinging nettle lectin) and hevein (Broakgert etal., 1989; Barkai-Golan et al., 1978). It is known that certain plantdiseases are caused by the production of phytotoxins. Resistance tothese diseases could be achieved through expression of a novel gene thatencodes an enzyme capable of degrading or otherwise inactivating thephytotoxin. Expression novel genes that alter the interactions betweenthe host plant and pathogen may be useful in reducing the ability thedisease organism to invade the tissues of the host plant, e.g., anincrease in the waxiness of the leaf cuticle or other morphologicalcharacteristics.

Plant parasitic nematodes are a cause of disease in many plants. It isproposed that it would be possible to make the plant resistant to theseorganisms through the expression of novel genes. It is anticipated thatcontrol of nematode infestations would be accomplished by altering theability of the nematode to recognize or attach to a host plant and/orenabling the plant to produce nematicidal compounds, including but notlimited to proteins.

5. Mycotoxin Reduction/Elimination

Production of mycotoxins, including aflatoxin and fumonisin, by fungiassociated with plants is a significant factor in rendering the grainnot useful. These fungal organisms do not cause disease symptoms and/orinterfere with the growth of the plant, but they produce chemicals(mycotoxins) that are toxic to animals. Inhibition of the growth ofthese fungi would reduce the synthesis of these toxic substances and,therefore, reduce grain losses due to mycotoxin contamination. Novelgenes may be introduced into plants that would inhibit synthesis of themycotoxin without interfering with fungal growth. Expression of a novelgene which encodes an enzyme capable of rendering the mycotoxin nontoxicwould be useful in order to achieve reduced mycotoxin contamination ofgrain. The result of any of the above mechanisms would be a reducedpresence of mycotoxins on grain.

6. Grain Composition or Quality

Genes may be introduced into plants, particularly commercially importantcereals such as maize, wheat or rice, to improve the grain for which thecereal is primarily grown. A wide range of novel transgenic plantsproduced in this manner may be envisioned depending on the particularend use of the grain.

For example, the largest use of maize grain is for feed or food.Introduction of genes that alter the composition of the grain maygreatly enhance the feed or food value. The primary components of maizegrain are starch, protein, and oil. Each of these primary components ofmaize grain may be improved by altering its level or composition.Several examples may be mentioned for illustrative purposes but in noway provide an exhaustive list of possibilities.

The protein of many cereal grains is suboptimal for feed and foodpurposes especially when fed to pigs, poultry, and humans. The proteinis deficient in several amino acids that are essential in the diet ofthese species, requiring the addition of supplements to the grain.Limiting essential amino acids may include lysine, methionine,tryptophan, threonine, valine, arginine, and histidine. Some amino acidsbecome limiting only after the grain is supplemented with other inputsfor feed formulations. For example, when the grain is supplemented withsoybean meal to meet lysine requirements, methionine becomes limiting.The levels of these essential amino acids in seeds and grain may beelevated by mechanisms which include, but are not limited to, theintroduction of genes to increase the biosynthesis of the amino acids,decrease the degradation of the amino acids, increase the storage of theamino acids in proteins, or increase transport of the amino acids to theseeds or grain.

One mechanism for increasing the biosynthesis of the amino acids is tointroduce genes that deregulate the amino acid biosynthetic pathwayssuch that the plant can no longer adequately control the levels that areproduced. This may be done by deregulating or bypassing steps in theamino acid biosynthetic pathway which are normally regulated by levelsof the amino acid end product of the pathway. Examples include theintroduction of genes that encode deregulated versions of the enzymesaspartokinase or dihydrodipicolinic acid (DHDP)-synthase for increasinglysine and threonine production, and anthranilate synthase forincreasing tryptophan production. Reduction of the catabolism of theamino acids may be accomplished by introduction of DNA sequences thatreduce or eliminate the expression of genes encoding enzymes thatcatalyze steps in the catabolic pathways such as the enzymelysine-ketoglutarate reductase.

The protein composition of the grain may be altered to improve thebalance of amino acids in a variety of ways including elevatingexpression of native proteins, decreasing expression of those with poorcomposition, changing the composition of native proteins, or introducinggenes encoding entirely new proteins possessing superior composition.DNA may be introduced that decreases the expression of members of thezein family of storage proteins. This DNA may encode ribozymes orantisense sequences directed to impairing expression of zein proteins orexpression of regulators of zein expression such as the opaque-2 geneproduct. The protein composition of the grain may be modified throughthe phenomenon of cosuppression, i.e., inhibition of expression of anendogenous gene through the expression of an identical structural geneor gene fragment introduced through transformation (Goring et al.,1991). Additionally, the introduced DNA may encode enzymes which degradeseines. The decreases in zein expression that are achieved may beaccompanied by increases in proteins with more desirable amino acidcomposition or increases in other major seed constituents such asstarch. Alternatively, a chimeric gene may be introduced that comprisesa coding sequence for a native protein of adequate amino acidcomposition such as for one of the globulin proteins or 10 kD zein ofmaize and a promoter or other regulatory sequence designed to elevateexpression of said protein. The coding sequence of said gene may includeadditional or replacement codons for essential amino acids. Further, acoding sequence obtained from another species, or, a partially orcompletely synthetic sequence encoding a completely unique peptidesequence designed to enhance the amino acid composition of the seed maybe employed.

The introduction of genes that alter the oil content of the grain may beof value. Increases in oil content may result in increases inmetabolizable energy content and density of the seeds for uses in feedand food. The introduced genes may encode enzymes that remove or reducerate-limitations or regulated steps in fatty acid or lipid biosynthesis.Such genes may include, but are not limited to, those that encodeacetyl-CoA carboxylase, ACP-acyltransferase, beta-ketoacyl-ACP synthase,plus other well known fatty acid biosynthetic activities. Otherpossibilities are genes that encode proteins that do not possessenzymatic activity such as acyl carrier protein. Additional examplesinclude 2-acetyltransferase, oleosin pyruvate dehydrogenase complex,acetyl CoA synthetase, ATP citrate lyase, ADP-glucose pyrophosphorylaseand genes of the carnitine-CoA-acetyl-CoA shuttles. It is anticipatedthat expression of genes related to oil biosynthesis will be targeted tothe plastid, using a plastid transit peptide sequence and preferablyexpressed in the seed embryo. Genes may be introduced that alter thebalance of fatty acids present in the oil providing a more healthful ornutritive feedstuff. The introduced DNA may also encode sequences thatblock expression of enzymes involved in fatty acid biosynthesis,altering the proportions of fatty acids present in the grain such asdescribed below.

Genes may be introduced that enhance the nutritive value of the starchcomponent of the grain, for example by increasing the degree ofbranching, resulting in improved utilization of the starch in cows bydelaying its metabolism.

Besides affecting the major constituents of the grain, genes may beintroduced that affect a variety of other nutritive, processing, orother quality aspects of the grain as used for feed or food. Forexample, pigmentation of the grain may be increased or decreased.Enhancement and stability of yellow pigmentation is desirable in someanimal feeds and may be achieved by introduction of genes that result inenhanced production of xanthophylls and carotenes by eliminatingrate-limiting steps in their production. Such genes may encode alteredforms of the enzymes phytoene synthase, phytoene desaturase, or lycopenesynthase. Alternatively, unpigmented white corn is desirable forproduction of many food products and may be produced by the introductionof DNA which blocks or eliminates steps in pigment production pathways.

Feed or food comprising some cereal grains possesses insufficientquantities of vitamins and must be supplemented to provide adequatenutritive value. Introduction of genes that enhance vitamin biosynthesisin seeds may be envisioned including, for example, vitamins A, E, B₁₂,choline, and the like. For example, maize grain also does not possesssufficient mineral content for optimal nutritive value. Genes thataffect the accumulation or availability of compounds containingphosphorus, sulfur, calcium, manganese, zinc, and iron among otherswould be valuable. An example may be the introduction of a gene thatreduced phytic acid production or encoded the enzyme phytase whichenhances phytic acid breakdown. These genes would increase levels ofavailable phosphate in the diet, reducing the need for supplementationwith mineral phosphate.

Numerous other examples of improvement of cereals for feed and foodpurposes might be described. The improvements may not even necessarilyinvolve the grain, but may, for example, improve the value of the grainfor silage. Introduction of DNA to accomplish this might includesequences that alter lignin production such as those that result in the“brown midrib” phenotype associated with superior feed value for cattle.

In addition to direct improvements in feed or food value, genes may alsobe introduced which improve the processing of grain and improve thevalue of the products resulting from the processing. The primary methodof processing certain grains such as maize is via wetmilling. Maize maybe improved though the expression of novel genes that increase theefficiency and reduce the cost of processing such as by decreasingsteeping time.

Improving the value of wetmilling products may include altering thequantity or quality of starch, oil, corn gluten meal, or the componentsof corn gluten feed. Elevation of starch may be achieved through theidentification and elimination of rate limiting steps in starchbiosynthesis or by decreasing levels of the other components of thegrain resulting in proportional increases in starch. An example of theformer may be the introduction of genes encoding ADP-glucosepyrophosphorylase enzymes with altered regulatory activity or which areexpressed at higher level. Examples of the latter may include selectiveinhibitors of, for example, protein or oil biosynthesis expressed duringlater stages of kernel development.

The properties of starch may be beneficially altered by changing theratio of amylose to amylopectin, the size of the starch molecules, ortheir branching pattern. Through these changes a broad range ofproperties may be modified which include, but are not limited to,changes in gelatinization temperature, heat of gelatinization, clarityof films and pastes, Theological properties, and the like. To accomplishthese changes in properties, genes that encode granule-bound or solublestarch synthase activity or branching enzyme activity may be introducedalone or combination. DNA such as antisense constructs may also be usedto decrease levels of endogenous activity of these enzymes. Theintroduced genes or constructs may possess regulatory sequences thattime their expression to specific intervals in starch biosynthesis andstarch granule development. Furthermore, it may be advisable tointroduce and express genes that result in the in vivo derivatization,or other modification, of the glucose moieties of the starch molecule.The covalent attachment of any molecule may be envisioned, limited onlyby the existence of enzymes that catalyze the derivatizations and theaccessibility of appropriate substrates in the starch granule. Examplesof important derivations may include the addition of functional groupssuch as amines, carboxyls, or phosphate groups which provide sites forsubsequent in vitro derivatizations or affect starch properties throughthe introduction of ionic charges. Examples of other modifications mayinclude direct changes of the glucose units such as loss of hydroxylgroups or their oxidation to aldehyde or carboxyl groups.

Oil is another product of wetmilling of corn and other grains, the valueof which may be improved by introduction and expression of genes. Thequantity of oil that can be extracted by wetmilling may be elevated byapproaches as described for feed and food above. Oil properties may alsobe altered to improve its performance in the production and use ofcooking oil, shortenings, lubricants or other oil-derived products orimprovement of its health attributes when used in the food-relatedapplications. Novel fatty acids may also be synthesized which uponextraction can serve as starting materials for chemical syntheses. Thechanges in oil properties may be achieved by altering the type, level,or lipid arrangement of the fatty acids present in the oil. This in turnmay be accomplished by the addition of genes that encode enzymes thatcatalyze the synthesis of novel fatty acids and the lipids possessingthem or by increasing levels of native fatty acids while possiblyreducing levels of precursors. Alternatively DNA sequences may beintroduced which slow or block steps in fatty acid biosynthesisresulting in the increase in precursor fatty acid intermediates. Genesthat might be added include desaturases, epoxidases, hydratases,dehydratases, and other enzymes that catalyze reactions involving fattyacid intermediates. Representative examples of catalytic steps thatmight be blocked include the desaturations from stearic to oleic acidand oleic to linolenic acid resulting in the respective accumulations ofstearic and oleic acids.

Improvements in the other major cereal wetmilling products, gluten mealand gluten feed, may also be achieved by the introduction of genes toobtain novel plants. Representative possibilities include but are notlimited to those described above for improvement of food and feed value.

In addition it may further be considered that the plant be used for theproduction or manufacturing of useful biological compounds that wereeither not produced at all, or not produced at the same level, in theplant previously. The novel plants producing these compounds are madepossible by the introduction and expression of genes by transformationmethods. The possibilities include, but are not limited to, anybiological compound which is presently produced by any organism such asproteins, nucleic acids, primary and intermediary metabolites,carbohydrate polymers, etc. The compounds may be produced by the plant,extracted upon harvest and/or processing, and used for any presentlyrecognized useful purpose such as pharmaceuticals, fragrances,industrial enzymes to name a few.

Further possibilities to exemplify the range of grain traits orproperties potentially encoded by introduced genes in transgenic plantsinclude grain with less breakage susceptibility for export purposes orlarger grit size when processed by dry milling through introduction ofgenes that enhance gamma-zein synthesis, popcorn with improved poppingquality and expansion volume through genes that increase pericarpthickness, corn with whiter grain for food uses though introduction ofgenes that effectively block expression of enzymes involved in pigmentproduction pathways, and improved quality of alcoholic beverages orsweet corn through introduction of genes which affect flavor such as theshrunken gene (encoding sucrose synthase) for sweet corn.

7. Plant Agronomic Characteristics

Two of the factors determining where plants can be grown are the averagedaily temperature during the growing season and the length of timebetween frosts. Within the areas where it is possible to grow aparticular plant, there are varying limitations on the maximal time itis allowed to grow to maturity and be harvested. The plant to be grownin a particular area is selected for its ability to mature and dry downto harvestable moisture content within the required period of time withmaximum possible yield. Therefore, plant of varying maturities aredeveloped for different growing locations. Apart from the need to drydown sufficiently to permit harvest is the desirability of havingmaximal drying take place in the field to minimize the amount of energyrequired for additional drying post-harvest. Also the more readily thegrain can dry down, the more time there is available for growth andkernel fill. Genes that influence maturity and/or dry down can beidentified and introduced into plant lines using transformationtechniques to create new varieties adapted to different growinglocations or the same growing location but having improved yield tomoisture ratio at harvest. Expression of genes that are involved inregulation of plant development may be especially useful, e.g., theliguleless and rough sheath genes that have been identified in plants.

Genes may be introduced into plants that would improve standability andother plant growth characteristics. For example, expression of novelgenes which confer stronger stalks, improved root systems, or prevent orreduce ear droppage would be of great value to the corn farmer.Introduction and expression of genes that increase the total amount ofphotoassimilate available by, for example, increasing light distributionand/or interception would be advantageous. In addition the expression ofgenes that increase the efficiency of photosynthesis and/or the leafcanopy would further increase gains in productivity. Such approacheswould allow for increased plant populations in the field.

Delay of late season vegetative senescence would increase the flow ofassimilate into the grain and thus increase yield. Overexpression ofgenes within plants that are associated with “stay green” or theexpression of any gene that delays senescence would achieve beadvantageous. For example, a non-yellowing mutant has been identified inFestuca pratensis (Davies et al., 1990). Expression of this gene as wellas others may prevent premature breakdown of chlorophyll and thusmaintain canopy function.

8. Nutrient Utilization

The ability to utilize available nutrients and minerals may be alimiting factor in growth of many plants. It is proposed that it wouldbe possible to alter nutrient uptake, tolerate pH extremes, mobilizationthrough the plant, storage pools, and availability for metabolicactivities by the introduction of novel genes. These modifications wouldallow a plant to more efficiently utilize available nutrients. It iscontemplated that an increase in the activity of, for example, an enzymethat is normally present in the plant and involved in nutrientutilization would increase the availability of a nutrient. An example ofsuch an enzyme would be phytase. It is also contemplated that expressionof a novel gene may make a nutrient source available that was previouslynot accessible, e.g., an enzyme that releases a component of nutrientvalue from a more complex molecule, perhaps a macromolecule.

9. Male Sterility

Male sterility is useful in the production of hybrid seed. It isproposed that male sterility may be produced through expression of novelgenes. For example, it has been shown that expression of genes thatencode proteins that interfere with development of the maleinflorescence and/or gametophyte result in male sterility. Chimericribonuclease genes that express in the anthers of transgenic tobacco andoilseed rape have been demonstrated to lead to male sterility (Marianiet al, 1990).

For example, a number of mutations were discovered in maize that confercytoplasmic male sterility. One mutation in particular, referred to as Tcytoplasm, also correlates with sensitivity to Southern corn leafblight. A DNA sequence, designated TURF-13 (Levings, 1990), wasidentified that correlates with T cytoplasm. It would be possiblethrough the introduction of TURF-13 via transformation to separate malesterility from disease sensitivity. As it is necessary to be able torestore male fertility for breeding purposes and for grain production,it is proposed that genes encoding restoration of male fertility mayalso be introduced.

10. Negative Selectable Markers

Introduction of genes encoding traits that can be selected against maybe useful for eliminating undesirable linked genes. When two or moregenes are introduced together by cotransformation, the genes will belinked together on the host chromosome. For example, a gene encoding aBt gene that confers insect resistance on the plant may be introducedinto a plant together with a bar gene that is useful as a selectablemarker and confers resistance to the herbicide Ignite® on the plant.However, it may not be desirable to have an insect resistant plant thatis also resistant to the herbicide Ignite®. It is proposed that onecould also introduce an antisense bar gene that is expressed in thosetissues where one does not want expression of the bar gene, e.g., inwhole plant parts. Hence, although the bar gene is expressed and isuseful as a selectable marker, it is not useful to confer herbicideresistance on the whole plant. The bar antisense gene is a negativeselectable marker.

Negative selection is necessary in order to screen a population oftransformants for rare homologous recombinants generated through genetargeting. For example, a homologous recombinant may be identifiedthrough the inactivation of a gene that was previously expressed in thatcell. The antisense gene to neomycin phosphotransferase II (nptII) hasbeen investigated as a negative selectable marker in tobacco (Nicotianatabacum) and Arabidopsis thaliana (Xiang and Guerra, 1993). In thisexample both sense and antisense nptII genes are introduced into a plantthrough transformation and the resultant plants arc sensitive to (ticantibiotic kanamycin. An introduced gene that integrates into the hostcell chromosome at the site of the antisense nptII gene, and inactivatesthe antisense gene, will make the plant resistant to kanamycin and otheraminoglycoside antibiotics. Therefore, rare site specific recombinantsmay be identified by screening for antibiotic resistance. Similarly, anygene, native to the plant or introduced through transformation, thatwhen inactivated confers resistance to a compound, may be useful as anegative selectable marker.

It is contemplated that negative selectable markers may also be usefulin other ways. One application is to construct transgenic lines in whichone could select for transposition to unlinked sites. In the process oftagging it is most common for the transposable element to move to agenetically linked site on the same chromosome. A selectable marker forrecovery of rare plants in which transposition has occurred to anunlinked locus would be useful. For example, the enzyme cytosinedeaminase may be useful for this purpose (Stouggard, 1993). In thepresence of this enzyme the compound 5-fluorocytosine is converted to5-fluoruracil which is toxic to plant and animal cells. If atransposable element is linked to the gene for the enzyme cytosinedeaminase, one may select for transposition to unlinked sites byselecting for transposition events in which the resultant plant is nowresistant to 5-fluorocytosine. The parental plants and plants containingtranspositions to linked sites will remain sensitive to5-fluorocytosine. Resistance to 5-fluorocytosine is due to loss of thecytosine deaminase gene through genetic segregation of the transposableelement and the cytosine deaminase gene. Other genes that encodeproteins that render the plant sensitive to a certain compound will alsobe useful in this context. For example, T-DNA gene 2 from Agrobacteriumtumefaciens encodes a protein that catalyzes the conversion ofalpha-naphthalene acetamide (NAM) to alpha-napthalene acetic acid (NAA)renders plant cells sensitive to high concentrations of NAM (Depicker etal., 1988).

It is also contemplated that negative selectable markers may be usefulin the construction of transposon tagging lines. For example, by markingan autonomous transposable element such as Ac, Master Mu, or En/Spn witha negative selectable marker, one could select for transformants inwhich the autonomous element is not stably integrated into the genome.This would be desirable, for example, when transient expression of theautonomous element is desired to activate in trans the transposition ofa defective transposable element, such as Ds, but stable integration ofthe autonomous element is not desired. The presence of the autonomouselement may not be desired in order to stabilize the defective element,i.e., prevent it from further transposing. However, it is proposed thatif stable integration of an autonomous transposable element is desiredin a plant the presence of a negative selectable marker may make itpossible to eliminate the autonomous element during the breedingprocess.

11. Non-Protein-Expressing Sequences

a. RNA-Expressing

DNA may be introduced into plants for the purpose of expressing RNAtranscripts that function to affect plant phenotype yet are nottranslated into protein. Two examples are antisense RNA and RNA withribozyme activity. Both may serve possible functions in reducing oreliminating expression of native or introduced plant genes.

Genes may be constructed or isolated, which when transcribed, produceantisense RNA that is complementary to all or part(s) of a targetedmessenger RNA(s). The antisense RNA reduces production of thepolypeptide product of the messenger RNA. The polypeptide product may beany protein encoded by the plant genome. The aforementioned genes willbe referred to as antisense genes. An antisense gene may thus beintroduced into a plant by transformation methods to produce a noveltransgenic plant with reduced expression of a selected protein ofinterest. For example, the protein may be an enzyme that catalyzes areaction in the plant. Reduction of the enzyme activity may reduce oreliminate products of the reaction which include any enzymaticallysynthesized compound in the plant such as fatty acids, amino acids,carbohydrates, nucleic acids and the like. Alternatively, the proteinmay be a storage protein, such as a zein, or a structural protein, thedecreased expression of which may lead to changes in seed amino acidcomposition or plant morphological changes respectively. Thepossibilities cited above are provided only by way of example and do notrepresent the full range of applications.

Genes may also be constructed or isolated, which when transcribedproduce RNA enzymes, or ribozymes, which can act as endoribonucleasesand catalyze the cleavage of RNA molecules with selected sequences. Thecleavage of selected messenger RNA's can result in the reducedproduction of their encoded polypeptide products. These genes may beused to prepare novel transgenic plants which possess them. Thetransgenic plants may possess reduced levels of polypeptides includingbut not limited to the polypeptides cited above that may be affected byantisense RNA. It is also possible that genes may be introduced toproduce novel transgenic plants which have reduced expression of anative gene product by a mechanism of cosuppression. It has beendemonstrated in tobacco, tomato, and petunia (Goring et al, 1991; Smithet al., 1990; Napoli et al., 1990; van der Krol et al., 1990) thatexpression of the sense transcript of a native gene will reduce oreliminate expression of the native gene in a manner similar to thatobserved for antisense genes. The introduced gene may encode all or partof the targeted native protein but its translation may not be requiredfor reduction of levels of that native protein.

b. Non-RNA-Expressing

For example, DNA elements including those of transposable elements suchas Ds, Ac, or Mu, may be inserted into a gene and cause mutations. TheseDNA elements may be inserted in order to inactivate (or activate) a geneand thereby “tag” a particular trail. In this instance the transposableelement does not cause instability of the tagged mutation, because theutility of the element does not depend on its ability to move in thegenome. Once a desired trait is tagged, the introduced DNA sequence maybe used to clone the corresponding gene, e.g., using the introduced DNAsequence as a PCR primer together with PCR gene cloning techniques(Shapiro, 1983; Dellaporta et al., 1988). Once identified, the entiregene(s) for the particular trait, including control or regulatoryregions where desired may be isolated, cloned and manipulated asdesired. The utility of DNA elements introduced into an organism forpurposed of gene tagging is independent of the DNA sequence and does notdepend on any biological activity of the DNA sequence, i.e.,transcription into RNA or translation into protein. The sole function ofthe DNA element is to disrupt the DNA sequence of a gene.

It is contemplated that unexpressed DNA sequences, including novelsynthetic sequences could be introduced into cells as proprietary“labels” of those cells and plants and seeds thereof. It would not benecessary for a label DNA element to disrupt the function of a geneendogenous to the host organism, as the sole function of this DNA wouldbe to identify the origin of the organism. For example, one couldintroduce a unique DNA sequence into a plant and this DNA element wouldidentify all cells, plants, and progeny of these cells as having arisenfrom that labeled source. It is proposed that inclusion of label DNAswould enable one to distinguish proprietary germplasm or germplasmderived from such, from unlabelled germplasm Another possible elementwhich may be introduced is a matrix attachment region element (MAR),such as the chicken lysozyme A element (Stief et al., 1989), which canbe positioned around an expressible gene of interest to effect anincrease in overall expression of the gene and diminish positiondependant effects upon incorporation into the plant genome (Stief etal., 1989; Phi-Van et al., 1990).

IV. Transformed (Transgenic) Plants of the Invention and Methods ofPreparation

Plant species may be transformed with the DNA construct of the presentinvention by the DNA-mediated transformation of plant cell protoplastsand subsequent regeneration of the plant from the transformedprotoplasts in accordance with procedures well known in the art.

Any plant tissue capable of subsequent clonal propagation, whether byorganogenesis or embryogenesis, may be transformed with a vector of thepresent invention. The term “organogenesis,” as used herein, means aprocess by which shoots and roots are developed sequentially frommeristematic centers; the term “embryogenesis,” as used herein, means aprocess by which shoots and roots develop together in a concertedfashion (not sequentially), whether from somatic cells or gametes. Theparticular tissue chosen will vary depending on the clonal propagationsystems available for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, megagametophytes, callus tissue,existing meristematic tissue (e.g., apical meristems, axillary buds, androot meristems), and induced meristem tissue (e.g., cotyledon meristemand ultilane meristem).

Plants of the present invent ion may take a variety of forms. The plantsmay be chimeras of transformed cells and non-transformed cells; theplants may be clonal transformants (e.g., all cells transformed tocontain the expression cassette); the plants may comprise grafts oftransformed and untransformed tissues (e.g., a transformed root stockgrafted to an untransformed scion in citrus species). The transformedplants may be propagated by a variety of means, such as by clonalpropagation or classical breeding techniques. For example, firstgeneration (or T1) transformed plants may be selfed to give homozygoussecond generation (or T2) transformed plants, and the T2 plants furtherpropagated through classical breeding techniques. A dominant selectablemarker (such as npt II) can be associated with the expression cassetteto assist in breeding.

Thus, the present invention provides a transformed (transgenic) plantcell, in planta or ex planta, including a transformed plastid or otherorganelle, e.g., nucleus, mitochondria or chloroplast. The presentinvention may be used for transformation of any plant species,including, but not limited to, corn (Zea mays), Brassica sp. (e.g., B.napus, B. rapa, B. juncea), particularly those Brassica species usefulas sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa),rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet(e.g., pearl millet (Pennisetum glaucum), proso millet (Panicummiliaceum), foxtail millet (Setaria italica), finger millet (Eleusinecoracana)), sunflower (Helianthus annuus), safflower (Carthamustinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco(Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachishypogaea) cotton (Gossypium barbadense, Gossypium hirsutum), sweetpotato (Ipomoea batatus), cassaya (Manihot esculenta), coffee (Cofeaspp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrustrees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis),banana (Musa spp.), avocado (Persea americana), fig (Ficus casica),guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamida (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables, ornamentals, and conifers; duckweed (Lemna, see WO 00/07210, which includes members of the family Lemnaceae.

There are known four genera and 34 species of duckweed as follows: genusLemna (L. aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L.japonica, L. minor, L. miniscula, L. obscura, L. perpusilla, L. tenera,L. trisulca, L. turionifera, L. valdiviana); genus Spirodela (S.intermedia, S. polyrrhiza, S. punctata); genus Woffia (Wa. angusta, Wa.arrhiza, Wa. australina, Wa. borealis, Wa. brasiliensis, Wa. columbiana,Wa. elongata, Wa. globosa, Wa. microscopica, Wa. neglecta) and genusWofiella (Wl. caudata, Wl. denticulata, Wl. gladiata, Wl. hyalina, Wl.lingulata, Wl. repunda, Wl. rotunda, and Wl. neotropica). Any othergenera or species of Lemnaceae, if they exist, are also aspects of thepresent invention. Lemna gibba, Lemna minor, and Lemna miniscula arepreferred, with Lemna minor and Lemna miniscula being most preferred.Lemna species can be classified using the taxonomic scheme described byLandolt, Biosystematic Investigation on the Family of Duckweeds: Thefamily of Lemnaceae—A Monograph Study. Geobotanisches Institut ETH,Stiftung Rubel, Zurich (1986)); vegetables including tomatoes(Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans(Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrusspp.), and members of the genus Cucumis such as cucumber (C. sativus),cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentalsinclude azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea),hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipaspp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation(Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), andchrysanthemum. Conifers that may be employed in practicing the presentinvention include, for example, pines such as loblolly pine (Pinustaeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa),lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata),Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis);Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firssuch as silver fir (Abies amabilis) and balsam fir (Abies balsamea); andcedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis); and leguminous plants.

Leguminous plants include beans and peas. Beans include guar, locustbean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean,fava bean, lentils, chickpea, etc. Legumes include, but are not limitedto, Arachis, e.g., peanuts, Vicia, e.g., crown vetch, hairy vetch,adzuki bean, mung bean, and chickpea, Lupinus, e.g., lupine, trifolium,Phaseolus, e.g., common bean and lima bean, Pisum, e.g., field bean,Melilotus, e.g., clover, Medicago, e.g., alfalfa, Lotus, e.g., trefoil,lens, e.g., lentil, and false indigo. Other sources for thepolynucleotides of the invention include Acacia, aneth, artichoke,arugula, blackberry, canola, cilantro, clementines, escarole,eucalyplus, fennel, grapefruit, honey dew, jicama, kiwifruit, lemon,lime, mushroom, nut, okra, orange, parsley, persimmon, plantain,pomegranate, poplar, radiata pine, radicchio, Southern pine, sweetgum,tangerine, triticale, vine, yams, apple, pear, quince, cherry, apricot,melon, hemp, buckwheat, grape, raspberry, chenopodium, blueberry,nectarine, peach, plum, strawberry, watermelon, eggplant, pepper,cauliflower, Brassica, e.g., broccoli, cabbage, brussel sprouts, onion,carrot, leek, beet, broad bean, celery, radish, pumpkin, endive, gourd,garlic, snapbean, spinach, squash, turnip, asparagus, and zucchini andornamental plants include impatiens, Begonia, Pelargonium, Viola,Cyclamen, Verbena, Vinca, Tagetes, Primula, Saint Paulia, Agertum,Amaranthus, Antihirrhinum, Aquilegia, Cineraria, Clover, Cosmo, Cowpea,Dahlia, Datura, Delphinium, Gerbera, Gladiolus, Gloxinia, Hippeastrum,Mesembryanthemum, Salpiglossos, and Zinnia.

Other plants for transformation include Cucurbitaceae, e.g., Cucumissativus, Cucumis melo, Citrullus lanatus, Cucurbita pepo, Cucurbitamaxima, and Cucurbita moschata; Solanaceae, e.g., Lycopersiconesculentum, Capsicum annuum, Capsicum frutescens, Solanum melongena,Nicotiana tabacum, Solanum tuberosum, Petunia×hybrida hort. ex E. Vilm.Brassicaceae, e.g., Brassica oleracea L. var. italica, Brassica oleraceaL var. capitata, Brassica rapa, Brassica oleracea L. var. botrytis,Raphanus sativus var. niger, and Brassica napus; Umbelliferae, e.g.,Daucus carota, Compositae, e.g., Lactuca sativa, and Helianthus annuus;Chenopodiaceae, e.g., Spinacia oleracea and Beta vulgaris; Leguminosae,e.g., Phaseolus vulgaris, Pisum sativum, and Glycine max; Gramineae,e.g., Zea mays; or Liliaceae, e.g., Allium cepa.

Preferred forage and turf grass plants for transformation includealfalfa, orchard grass, tall fescue, perennial ryegrass, creeping bentgrass, and redtop. Preferably, the plants are crop plants and inparticular cereals, e.g., wheat, rice, corn, oats, barley, and rye, andeven more preferably rice, corn and wheat.

Transformation of plants can be undertaken with a single DNA molecule ormultiple DNA molecules (i.e., co-transformation), and both thesetechniques are suitable for use with the expression cassettes of thepresent invention. Numerous transformation vectors are available forplant transformation, and the expression cassettes of this invention canbe used in conjunction with any such vectors. The selection of vectorwill depend upon the preferred transformation technique and the targetspecies for transformation.

A variety of techniques are available and known to those skilled in theart for introduction of constructs into a plant cell host. Thesetechniques generally include transformation with DNA employing A.tunefaciens or A. rhizogenes as the transforming agent, liposomes, PEGprecipitation, electroporation, DNA injection, direct DNA uptake,microprojectile bombardment, particle acceleration, and the like (See,for example, EP 295959 and EP 138341) (see below). However, cells otherthan plant cells may be transformed with the expression cassettes of theinvention. The general descriptions of plant expression vectors andreporter genes, and Agrobacterium and Agrobacterium-mediated genetransfer, can be found in Gruber et al., (1993).

Expression vectors containing genomic or synthetic fragments can beintroduced into protoplasts or into intact tissues or isolated cells.Preferably expression vectors are introduced into intact tissue. Generalmethods of culturing plant tissues are provided for example by Maki etal., (1993); and by Phillips et al., (1988). Preferably, expressionvectors are introduced into maize or other plant tissues using a directgene transfer method such as microprojectile-mediated delivery, DNAinjection, electroporation and the like. More preferably expressionvectors are introduced into plant tissues using the microprojectilemedia delivery with the biolistic device. See, for example, Tomes et al.(1995). The vectors of the invention can not only be used for expressionof structural genes but may also be used in exon-trap cloning, orpromoter trap procedures to detect differential gene expression invarieties of tissues, (Lindsey et al., 1993; Auch & Reth et al.).

It is particularly preferred to use the binary type vectors of Ti and R1plasmids of Agrobacterium spp. Ti-derived vectors transform a widevariety of higher plants, including monocotyledonous and dicotyledonousplants, such as soybean, cotton, rape, tobacco, and rice (Pacciotti etal., 1985: Byrne et al., 1987; Sukhapinda et al., 1987; Park et al.,1985: Hiei et al., 1994). The use of T-DNA to transform plant cells hasreceived extensive study and is amply described (EP 120516; Hoekema,1985; Knauf, et al., 1983; and An et al., 1985). For introduction intoplants, the chimeric genes of the invention can be inserted into binaryvectors as described in the examples.

Other transformation methods are available to those skilled in the art,such as direct uptake of foreign DNA constructs (see EP 295959),techniques of electroporation (Fromm et al., 1986) or high velocityballistic bombardment with metal particles coated with the nucleic acidconstructs (Kline et al., 1987, and U.S. Pat. No. 4,945,050). Oncetransformed, the cells can be regenerated by those skilled in the art.Of particular relevance are the recently described methods to transformforeign genes into commercially important crops, such as rapeseed (DeBlock et al., 1989), sunflower (Everett et al., 1987), soybean (McCabeet al., 1988; Hinchee et al., 1988; Chee et al., 1989; Christou et al.,1989; EP 301749), rice (Hiei et al., 1994), and corn (Gordon Kamm etal., 1990; Fromm et al., 1990).

Those skilled in the art will appreciate that the choice of method mightdepend on the type of plant, i.e., monocotyledonous or dicotyledonous,targeted for transformation. Suitable methods of transforming plantcells include, but are not limited to, microinjection (Crossway et al.,1986), electroporation (Riggs et al., 1986), Agrobacterium-mediatedtransformation (Hinchee et al., 1988), direct gene transfer (Paszkowskiet al., 1984), and ballistic particle acceleration using devicesavailable from Agracetus, Inc., Madison, Wis. And BioRad, Hercules,Calif. (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; andMcCabe et al., 1988). Also see, Weissinger et al., 1988; Sanford et al.,1987 (onion); Christou et al., 1988 (soybean); McCabe et al., 1988(soybean); Datta et al., 1990 (rice); Klein et al., 1988 (maize); Kleinet al., 1988 (maize); Klein et al., 1988 (maize); Fromm et al., 1990(maize); and Gordon-Kamm et al., 1990 (maize); Svab et al., 1990(tobacco chloroplast); Koziel et al., 1993 (maize); Shimamoto et al.,1989 (rice); Christou et al., 1991 (rice); European Patent ApplicationEP 0 332 581 (orchardgrass and other Pooideae); Vasil et al., 1993(wheat); Weeks et al., 1993 (wheat). In one embodiment, the protoplasttransformation method for maize is employed (European Patent ApplicationEP 0 292 435, U.S. Pat. No. 5,350,689).

In another embodiment, a nucleotide sequence of the present invention isdirectly transformed into the plastid genome. Plastid transformationtechnology is extensively described in U.S. Pat. Nos. 5,451,513,5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and inMcBride et al., 1994. The basic technique for chloroplast transformationinvolves introducing regions of cloned plastid DNA flanking a selectablemarker together with the gene of interest into a suitable target tissue,e.g., using biolistics or protoplast transformation (e.g., calciumchloride or PEG mediated transformation). The 1 to 1.5 kb flankingregions, termed targeting sequences, facilitate orthologousrecombination with the plastid genome and thus allow the replacement ormodification of specific regions of the plastome. Initially, pointmutations in the chloroplast 16S rRNA and rps12 genes conferringresistance to spectinomycin and/or streptomycin are utilized asselectable markers for transformation (Svab et al., 1990; Staub et al.,1992). This resulted in stable homoplasmic transformants at a frequencyof approximately one per 100 bombardments of target leaves. The presenceof cloning sites between these markers allowed creation of a plastidtargeting vector for introduction of foreign genes (Staub et al., 1993).Substantial increases in transformation frequency are obtained byreplacement of the recessive rRNA or r-protein antibiotic resistancegenes with a dominant selectable marker, the bacterial aadA geneencoding the spectinomycin-detoxifying enzymeaminoglycoside-3N-adenyltransferase (Svab et al., 1993). Otherselectable markers useful for plastid transformation are known in theart and encompassed within the scope of the invention. Typically,approximately 15–20 cell division cycles following transformation arerequired to reach a homoplastidic state. Plastid expression, in whichgenes are inserted by orthologous recombination into all of the severalthousand copies of the circular plastid genome present in each plantcell, takes advantage of the enormous copy number advantage overnuclear-expressed genes to permit expression levels that can readilyexceed 10% of the total soluble plant protein. In a preferredembodiment, a nucleotide sequence of the present invention is insertedinto a plastid targeting vector and transformed into the plastid genomeof a desired plant host. Plants homoplastic for plastid genomescontaining a nucleotide sequence of the present invention are obtained,and are preferentially capable of high expression of the nucleotidesequence.

Agrobacterium tumefaciens cells containing a vector comprising anexpression cassette of the present invention, wherein the vectorcomprises a Ti plasmid, are useful in methods of making transformedplants. Plant cells are infected with an Agrobacterium tumefaciens asdescribed above to produce a transformed plant cell, and then a plant isregenerated from the transformed plant cell. Numerous Agrobacteriumvector systems useful in carrying out the present invention are known.

For example, vectors are available for transformation usingAgrobacterium tumefaciens. These typically carry at least one T-DNAborder sequence and include vectors such as pBIN19 (Bevan, 1984). In onepreferred embodiment, the expression cassettes of the present inventionmay be inserted into either of the binary vectors pCIB200 and pCIB2001for use with Agrobacterium. These vector cassettes forAgrobacterium-mediated transformation wear constructed in the followingmanner. PTJS75kan was created by Narl digestion of pTJS75 (Schmidhauser& Helinski, 1985) allowing excision of the tetracycline-resistance gene,followed by insertion of an AccI fragment from pUC4K carrying an NPTII(Messing & Vierra, 1982; Bevan et al., 1983; McBride et al., 1990). XhoIlinkers were ligated to the EcoRV fragment of pCIB7 which contains theleft and right T-DNA borders, a plant selectable nos/nptII chimeric geneand the pUC polylinker (Rothstein et al., 1987), and the XhoI-digestedfragment was cloned into SalI-digested pTJS75kan to create pCIB200 (seealso EP 0 332 104, example 19). PCIB200 contains the following uniquepolylinker restriction sites: EcoRI, SstI KpnI, BgII, XbaI, and SalI.The plasmid pCIB2001 is a derivative of pCIB200 which was created by theinsertion into the polylinker of additional restriction sites. Uniquerestriction sites in the polylinker of pCIB2001 are EcoRI, Sstl, KpnI,BgIII, XbaI, SalI, Mlul, BclI, AvrII, ApaI, HpaI, and Stul. PCIB2001, inaddition to containing these unique restriction sites also has plant andbacterial kanamycin selection, left and right T-DNA borders forAgrobacterium-mediated transformation, the RK2-derived trfA function formobilization between E. coli and other hosts, and the CriT and OriVfunctions also from RK2. The pCIB2001 polylinker is suitable for thecloning of plant expression cassettes containing their own regulatorysignals.

An additional vector useful for Agrobacterium-mediated transformation isthe binary vector pCIB 10, which contains a gene encoding kanamycinresistance for selection in plants, T-DNA right and left bordersequences and incorporates sequences from the wide host-range plasmidpRK252 allowing it to replicate in both E. coli and Agrobacterium. Itsconstruction is described by Rothstein et al., 1987. Various derivativesof pCIB10 have been constructed which incorporate the gene forhygromycin B phosphotransferase described by Gritz et al., 1983. Thesederivatives enable selection of transgenic plant cells on hygromycinonly (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717).

Methods using either a form of direct gene transfer orAgrobacterium-mediated transfer usually, but not necessarily, areundertaken with a selectable marker which may provide resistance to anantibiotic (e.g., kanamycin, hygromycin or methotrexate) or a herbicide(e.g., phosphinothricin). The choice of selectable marker for planttransformation is not, however, critical to the invention.

For certain plant species, different antibiotic or herbicide selectionmarkers may be preferred. Selection markers used routinely intransformation include the nptlI gene which confers resistance tokanamycin and related antibiotics (Messing & Vierra, 1982; Bevan et al.,1983), the bar gene which confers resistance to the herbicidephosphinothricin (White et al., 1990, Spencer et al., 1990), the hphgene which confers resistance to the antibiotic hygromycin (Blochinger &Diggelmann), and the dhfr gene, which confers resistance to methotrexate(Bourouis et al., 1983).

One such vector useful for direct gene transfer techniques incombination with selection by the herbicide Basta (or phosphinothricin)is pCIB3064. This vector is based on the plasmid pCIB246, whichcomprises the CaMV 35S promoter in operational fusion to the E. coli GUSgene and the CaMV 35S transcriptional terminator and is described in thePCT published application WO 93/07278, herein incorporated by reference.One gene useful for conferring resistance to phosphinothricin is the bargene from Streptomyces viridochromogenes (Thompson et al., 1987). Thisvector is suitable for the cloning of plant expression cassettescontaining their own regulatory signals.

An additional transformation vector is pSOG35 which utilizes the E. coligene dihydrofolate reductase (DHFR) as a selectable marker conferringresistance to methotrexate. PCR was used to amplify the 35S promoter(about 800 bp), intron 6 from the maize Adhl gene (about 550 bp) and 18bp of the GUS untranslated leader sequence from pSOG10. A 250 bpfragment encoding the E. coli dihydrofolate reductase type 11 gene wasalso amplified by PCR and these two PCR fragments were assembled with aSacI-PstI fragment from pBI221 (Clontech) which comprised the pUC19vector backbone and the nopaline synthase terminator. Assembly of thesefragments generated pSOG19 which contains the 35S promoter in fusionwith the intron 6 sequence, the GUS leader, the DHFR gene and thenopaline synthase terminator. Replacement of the GUS leader in pSOG19with the leader sequence from Maize Chlorotic Mottle Virus check (MCMV)generated the vector pSOG35. pSOG19 and pSOG35 carry the pUC-derivedgene for ampicillin resistance and have HindIII, SphI, PstI and EcoRIsites available for the cloning of foreign sequences.

V. Production and Characterization of Stably Transformed Plants

Transgenic plant cells are then placed in an appropriate selectivemedium for selection of transgenic cells which are then grown to callus.Shoots are grown from callus and plantlets generated from the shoot bygrowing in rooting medium. The various constructs normally will bejoined to a marker for selection in plant cells. Conveniently. themarker may be resistance to a biocide (particularly an antibiotic, suchas kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicide,or the like). The particular marker used will allow for selection oftransformed cells as compared to cells lacking the DNA which has beenintroduced. Components of DNA constructs including transcriptioncassettes of this invention may be prepared from sequences which arenative (endogenous) or foreign (exogenous) to the host. By “foreign” itis meant that the sequence is not found in the wild-type host into whichthe construct is introduced. Heterologous constructs will contain atleast one region which is not native to the gene from which thetranscription-initiation-region is derived.

To confirm the presence of the transgenes in transgenic cells andplants, a variety of assays may be performed. Such assays include, forexample, “molecular biological” assays well known to those of skill inthe art, such as Southern and Northern blotting, in situ hybridizationand nucleic acid-based amplification methods such as PCR or RT-PCR;“biochemical” assays, such as detecting the presence of a proteinproduct, e.g., by immunological means (ELISAs and Western blots) or byenzymatic function; plant part assays, such as leaf or root assays; andalso, by analyzing the phenotype of the whole regenerated plant, e.g.,for disease or pest resistance.

DNA may be isolated from cell lines or any plant parts to determine thepresence of the preselected nucleic acid segment through the use oftechniques well known to those skilled in the art. Note that intactsequences will not always be present, presumably due to rearrangement ordeletion of sequences in the cell.

The presence of nucleic acid elements introduced through the methods ofthis invention may be determined by polymerase chain reaction (PCR).Using this technique discreet fragments of nucleic acid are amplifiedand detected by gel electrophoresis. This type of analysis permits oneto determine whether a preselected nucleic acid segment is present in astable transformant, but does not prove integration of the introducedpreselected nucleic acid segment into the host cell genome. In addition,it is not possible using PCR techniques to determine whethertransformants have exogenous genes introduced into different sites inthe genome, i.e., whether transformants are of independent origin. It iscontemplated that using PCR techniques it would be possible to clonefragments of the host genomic DNA adjacent to an introduced preselectedDNA segment.

Positive proof of DNA integration into the host genome and theindependent identities of transformants may be determined using thetechnique of Southern hybridization. Using this technique specific DNAsequences that were introduced into the host genome and flanking hostDNA sequences can be identified. Hence the Southern hybridizationpattern of a given transformant serves as an identifying characteristicof that transformant. In addition it is possible through Southernhybridization to demonstrate the presence of introduced preselected DNAsegments in high molecular weight DNA, i.e., confirm that the introducedpreselected DNA segment has been integrated into the host cell genome.The technique of Southern hybridization provides information that isobtained using PCR, e.g., the presence of a preselected DNA segment, butalso demonstrates integration into the genome and characterizes eachindividual transformant.

It is contemplated that using the techniques of dot or slot blothybridization which are modifications of Southern hybridizationtechniques one could obtain the same information that is derived fromPCR, e.g., the presence of a preselected DNA segment.

Both PCR and Southern hybridization techniques can be used todemonstrate transmission of a preselected DNA segment to progeny. Inmost instances the characteristic Southern hybridization pattern for agiven transformant will segregate in progeny as one or more Mendeliangenes (Spencer et al., 1992); Laursen et al., 1994) indicating stableinheritance of the gene. The nonchimeric nature of the callus and theparental transformants (R₀) was suggested by germline transmission andthe identical Southern blot hybridization patterns and intensities ofthe transforming DNA in callus, R₀ plants and R₁ progeny that segregatedfor the transformed gene.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA may only be expressed in particular cells ortissue types and hence it will be necessary to prepare RNA for analysisfrom these tissues. PCR techniques may also be used for detection andquantitation of RNA produced from introduced preselected DNA segments.In this application of PCR it is first necessary to reverse transcribeRNA into DNA, using enzymes such as reverse transcriptase, and thenthrough the use of conventional PCR techniques amplify the DNA. In mostinstances PCR techniques, while useful, will not demonstrate integrityof the RNA product. Further information about the nature of the RNAproduct may be obtained by Northern blotting. This technique willdemonstrate the presence of an RNA species and give information aboutthe integrity of that RNA. The presence or absence of an RNA species canalso be determined using dot or slot blot Northern hybridizations. Thesetechniques are modifications of Northern blotting and will onlydemonstrate the presence or absence of an RNA species.

While Southern blotting and PCR may be used to detect the preselectedDNA segment in question, they do not provide information as to whetherthe preselected DNA segment is being expressed. Expression may beevaluated by specifically identifying the protein products of theintroduced preselected DNA segments or evaluating the phenotypic changesbrought about by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focusing, or by chromatographictechniques such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins offer opportunities for use ofspecific antibodies to detect their presence in formats such as an ELISAassay. Combinations of approaches may be employed with even greaterspecificity such as Western blotting in which antibodies are used tolocate individual gene products that have been separated byelectrophoretic techniques. Additional techniques may be employed toabsolutely confirm the identity of the product of interest such asevaluation by amino acid sequencing following purification. Althoughthese are among the most commonly employed, other procedures may beadditionally used.

Assay procedures may also be used to identify the expression of proteinsby their functionality, especially the ability of enzymes to catalyzespecific chemical reactions involving specific substrates and products.These reactions may be followed by providing and quantifying the loss ofsubstrates or the generation of products of the reactions by physical orchemical procedures. Examples are as varied as the enzyme to beanalyzed.

Very frequently the expression of a gene product is determined byevaluating the phenotypic results of its expression. These assays alsomay take many forms including but not limited to analyzing changes inthe chemical composition, morphology, or physiological properties of theplant. Morphological changes may include greater stature or thickerstalks. Most often changes in response of plants or plant parts toimposed treatments are evaluated under carefully controlled conditionstermed bioassays.

VI. Uses of Transgenic Plants

Once an expression cassette of the invention has been transformed into aparticular plant species, it may be propagated in that species or movedinto other varieties of the same species, particularly includingcommercial varieties, using traditional breeding techniques.Particularly preferred plants of the invention include the agronomicallyimportant crops listed above. The genetic properties engineered into thetransgenic seeds and plants described above are passed on by sexualreproduction and can thus be maintained and propagated in progenyplants. The present invention also relates to a transgenic plant cell,tissue, organ, seed or plant part obtained from the transgenic plant.Also included within the invention are transgenic descendants of theplant as well as transgenic plant cells, tissues, organs, seeds andplant parts obtained from the descendants.

Preferably, the expression cassette in the transgenic plant is sexuallytransmitted. In one preferred embodiment, the coding sequence issexually transmitted through a complete normal sexual cycle of the R0plant to the R1 generation. Additionally preferred, the expressioncassette is expressed in the cells, tissues, seeds or plant of atransgenic plant in an amount that is different than the amount in thecells, tissues, seeds or plant of a plant which only differs in that theexpression cassette is absent.

The transgenic plants produced herein are thus expected to be useful fora variety of commercial and research purposes. Transgenic plants can becreated for use in traditional agriculture to possess traits beneficialto the grower (e.g., agronomic traits such as resistance to waterdeficit, pest resistance, herbicide resistance or increased yield),beneficial to the consumer of the grain harvested from the plant (e.g.,improved nutritive content in human food or animal feed; increasedvitamin, amino acid, and antioxidant content; the production ofantibodies (passive immunization) and nutriceuticals), or beneficial tothe food processor (e.g., improved processing traits). In such uses, theplants are generally grown for the use of their grain in human or animalfoods. Additionally, the use of root-specific promoters in transgenicplants can provide beneficial traits that are localized in theconsumable (by animals and humans) roots of plants such as carrots,parsnips, and beets. However, other parts of the plants, includingstalks, husks, vegetative parts, and the like, may also have utility,including use as part of animal silage or for ornamental purposes.Often, chemical constituents (e.g., oils or starches) of maize and othercrops are extracted for foods or industrial use and transgenic plantsmay be created which have enhanced or modified levels of suchcomponents.

Transgenic plants may also find use in the commercial manufacture ofproteins or other molecules, where the molecule of interest is extractedor purified from plant parts, seeds, and the like. Cells or tissue fromthe plants may also be cultured, grown in vitro, or fermented tomanufacture such molecules.

The transgenic plants may also be used in commercial breeding programs,or may be crossed or bred to plants of related crop species.Improvements encoded by the expression cassette may be transferred,e.g., from maize cells to cells of other species, e.g., by protoplastfusion.

The transgenic plants may have many uses in research or breeding,including creation of new mutant plants through insertional mutagenesis,in order to identify beneficial mutants that might later be created bytraditional mutation and selection. An example would be the introductionof a recombinant DNA sequence encoding a transposable element that maybe used for generating genetic variation. The methods of the inventionmay also be used to create plants having unique “signature sequences” orother marker sequences which can be used to identify proprietary linesor varieties.

Thus, the transgenic plants and seeds according to the invention can beused in plant breeding which aims at the development of plants withimproved properties conferred by the expression cassette, such astolerance of drought, disease, or other stresses. The various breedingsteps are characterized by well-defined human intervention such asselecting the lines to be crossed, directing pollination of the parentallines, or selecting appropriate descendant plants. Depending on thedesired properties different breeding measures are taken. The relevanttechniques are well known in the art and include but are not limited tohybridization, inbreeding, backcross breeding, ultilane breeding,variety blend, interspecific hybridization, aneuploid techniques, etc.Hybridization techniques also include the sterilization of plants toyield male or female sterile plants by mechanical, chemical orbiochemical means. Cross pollination of a male sterile plant with pollenof a different line assures that the genome of the male sterile butfemale fertile plant will uniformly obtain properties of both parentallines. Thus, the transgenic seeds and plants according to the inventioncan be used for the breeding of improved plant lines which for exampleincrease the effectiveness of conventional methods such as herbicide orpesticide treatment or allow to dispense with said methods due to theirmodified genetic properties. Alternatively new crops with improvedstress tolerance can be obtained which, due to their optimized genetic“equipment”, yield harvested product of better quality than productswhich were not able to tolerate comparable adverse developmentalconditions.

VII. A Computer Readable Medium

The invention also provides a computer readable medium having storedthereon a data structure containing nucleic acid sequences having atleast 70% sequence identity to SEQ ID NO:1, as well as complementary,ortholog, and variant sequences thereof. Storage and use of nucleic acidsequences on a computer readable medium is well known in the art. (Seefor example U.S. Pat. Nos. 6,023,659; 5,867,402; 5,795,716) Examples ofsuch medium include, but are not limited to, magnetic tape, opticaldisk, CD-ROM, random access memory, volatile memory, non-volatile memoryand bubble memory. Accordingly, the nucleic acid sequences contained onthe computer readable medium may be compared through use of a modulethat receives the sequence information and compares it to other sequenceinformation. Examples of other sequences to which the nucleic acidsequences of the invention may be compared include those maintained bythe National Center for Biotechnology Information (NCBI)(available onthe World Wide Web at ncbi.-nlm.nih.gov/) and the Swiss Protein DataBank. A computer is an example of such a module that can read andcompare nucleic acid sequence information. Accordingly, the inventionalso provides the method of comparing a nucleic acid sequence of theinvention to another sequence. For example, a sequence of the inventionmay be submitted to the NCBI for a Blast search as described hereinwhere the sequence is compared to sequence information contained withinthe NCBI database and a comparison is returned. The invention alsoprovides nucleic acid sequence information in a computer readable mediumthat allows the encoded polypeptide to be optimized for a desiredproperty. Examples of such properties include, but are not limited to,increased or decreased: thermal stability, chemical stability,hydrophylicity, hydrophobicity, and the like. Methods for the use ofcomputers to model polypeptides and polynucleotides having alteredactivities are well known in the art and have been reviewed. (Lesyng etal., 1993; Surles et al., 1994; Koehl et al., 1996; Rossi et al., 2001).

VIII. Inhibition of Expression of the Cap Binding Protein 80 Gene

A. Antisense Technology

A number of methods can be used to inhibit the expression of genesencoding cap binding protein 80 in plants. For instance, antisensetechnology can be conveniently used. To accomplish this, a nucleotidesequence from the desired gene is cloned and operably linked to apromoter such that the antisense strand of RNA is transcribed.Preferably, the promoter is one which drives expression specifically inleaf cells. More preferably, the promoter drives expression specificallyin guard cells. The construct is then transformed into plants and theantisense strand of RNA is produced. In plant cells, it has beensuggested that antisense suppression can act at all levels of generegulation including suppression of RNA translation (see, Bourque, 1995;Pantopoulos, 1989; Heiser et al. 1997) and by preventing theaccumulation of mRNA which encodes the protein of interest (see,Baulcombe, 1996; Prins and Goldbach, 1996; Metzlaff et al., 1997; Sheehyet al., 1988, and U.S. Pat. No. 4,801,340).

The nucleotide sequence to be introduced generally will be substantiallyidentical to at least a portion of the endogenous cap binding protein 80gene or other genes to be repressed. The sequence, however, need not beperfectly identical to inhibit expression. The constructs of the presentinvention can be designed such that the inhibitory effect applies toother genes within a family of genes exhibiting identity or substantialidentity to the target gene.

For antisense suppression, the introduced sequence also need not be fulllength relative to either the primary transcription product or fullyprocessed mRNA. Generally, higher identity can be used to compensate forthe use of a shorter sequence. Furthermore, the introduced sequence neednot have the same intron or exon pattern, and identity of non-codingsegments may be equally effective. Normally, a sequence of between about30 or 40 nucleotides and about the full-length sequence should be used,though a sequence of at least about 100 nucleotides is preferred, asequence of at least about 200 nucleotides is more preferred, and asequence of about 700 to about 3000 nucleotides is especially preferred.

A number of gene regions can be targeted to suppress cap binding protein80 gene expression. The targets can include, for instance, the codingregions, introns, sequences from exon/intron junctions, 5′ or3′untranslated regions, and the like.

Inhibiting the expression of cap binding protein 80 results ininhibition of the enzyme's activity. It may be desirable to inhibitexpression in a way which targets the inhibition to guard cells or toother selected cell types in which the inhibition of cap binding protein80 is desired. This can be done, for example, by using a promoter whichis preferentially or primarily expressed in the cell type of interest.

B. Co-suppression and Double Stranded RNA Gene Silencing

Several techniques have recently been developed for blocking orsuppressing the expression of endogenous genes by introduction ofexogenous nucleic acids. One well known method of suppression is senseco-suppression. Introduction of nucleic acid configured in the senseorientation has been recently shown to be an effective means by which toblock the transcription of target genes. For examples of the use of thismethod to modulate expression of endogenous genes see, for example,Assaad et al., 1993; Flavell, 1994; Stam et al., 1997 Napoli et al.,1990; and U.S. Pat. Nos. 5,034,323, 5,231,020, and 5,283,184.Post-transcriptional gene silencing induced by introduction oftransgenes blocks expression of endogenous genes homologous to thetransgene and is thought to be involved in virus resistance and genomemaintenance. See, e.g., Cogoni and Macino, 1992. The suppressive effectmay occur where the introduced sequence contains no coding sequence perse, but only intron or untranslated sequences homologous to sequencespresent in the primary transcript of the endogenous sequence.

The-introduced sequence generally will be substantially identical to theendogenous sequence intended to be repressed. This minimal identity willtypically be greater than about 65%, but a higher identity might exert amore effective repression of expression, e.g., about 95% to absoluteidentity is preferred. As with antisense regulation, the effect shouldapply to any other proteins within a similar family of genes exhibitingidentity or substantial identity.

For co-suppression, the introduced sequence, needing less than absoluteidentity, also need not be full length, relative to either the primarytranscription product or fully processed mRNA. This may be preferred toavoid concurrent production of some plants which are overexpressers. Ahigher identity in a shorter than full length sequence compensates for alonger, less identical sequence. Furthermore, the introduced sequenceneed not have the same intron or exon pattern, and identity ofnon-coding segments will be equally effective. Normally, a sequence ofthe size ranges noted above for antisense regulation is used. Inaddition, the same gene regions noted for antisense regulation can betargeted using co-suppression technologies.

In a further approach to blocking expression of endogenous genes, it hasbeen found that RNA capable of forming duplexes of sense and antisensestrands suppresses expression of endogenous genes more powerfully thandoes introduction of either strand individually in organisms as diverseas plants and worms. See, e.g., Waterhouse et al., 1998; Fire et al.,1998. Post-transcriptional gene silencing by introduction of doublestranded RNA therefore provides another powerful tool for inhibiting capbinding protein 80 activity.

C. Triplex DNA Formation

Oligonucleotide-based triple-helix formation can also be used to disruptcap binding protein 80 gene expression. Triplex DNA can inhibit DNAtranscription and replication, generate site-specific mutations, cleaveDNA, and induce homologous recombination (see, e.g., Havre and Glazer,1993; Scanlon et al., 1995; Giovannangeli et al., 1996; Chan and Glazer,1997. Triple helix DNAs can be used to target the same sequencesidentified for antisense regulation.

D. Ribozymes

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of cap binding protein 80 genes. It is possible to designribozymes that specifically pair with virtually any target RNA andcleave the phosphodiester backbone at a specific location, therebyfunctionally inactivating the target RNA. In carrying out this cleavage,the ribozyme is not itself altered, and is thus capable of recycling andcleaving other molecules, making it a true enzyme. The inclusion ofribozyme sequences within antisense RNAs confers RNA-cleaving activityupon them, thereby increasing the activity of the constructs. Thus,ribozymes can be used to target the same sequences identified forantisense regulation. In preferred embodiments, the ribozymes aretargeted to specific cell types, such as guard cells or leaf cells.Conveniently, this can be achieved by introducing a DNA construct with apromoter specific for the cell type of interest.

A number of classes of ribozymes have been identified. One class ofribozymes is derived from a number of small circular RNAs which arecapable of self cleavage and replication in plants. The RNAs replicateeither alone (viroid RNAs) or with a helper virus (satellite RNAs).Examples include RNAs from avocado sunblotch or viroid and the satelliteRNAs from tobacco ringspot virus, lucerne transient streak virus, velvettobacco mottle virus, solanum nodiflorum mottle virus and subterraneanclover mottle virus. The design and use of target RNA-specific ribozymesis described in Zhao and Pick, 1993 Eastham and Ahlering, 1996; Sokoland Murray, 1996; Sun et al., 1997; and Haseloff et al., 1988.

E. Modification and Knocking Out of Endogenous Genes

In addition to blocking expression of endogenous genes, as discussedabove, methods for introducing genetic mutations into the genes can alsobe used to select for plants with decreased cap binding protein 80expression. In preferred embodiments, endogenous genes encoding capbinding protein 80 are modified by, for example, replacing the promoterwith a promoter with lower activity, or with an inducible promoter sothat expression of the gene can be turned off when conditions warrant.

Methods for introducing genetic mutations into plant genes and selectingplants with desired traits are well known. For instance, seeds or otherplant material can be treated with a mutagenic chemical substance,according to standard techniques. Such chemical substances include, butare not limited to, the following: diethyl sulfate, ethylene imine,ethyl methanesulfonate and N-nitroso-N ethylurea. Alternatively,ionizing radiation from sources such as, X-rays or gamma rays can beused.

Alternatively, homologous recombination can be used to induce targetedgene modifications by specifically targeting the cap binding protein 80gene in vivo (see, generally, Grewal and Klar, 1997; Xu et al., 1996).Homologous recombination has been demonstrated in plants (Puchta et al.,1994, Swoboda et al., 1994; Offtinga et al., 1993; and Kempin et al.,1997).

In applying homologous recombination technology to cap binding protein80 genes, mutations in selected portions of a cap binding protein 80gene sequence (including 5′ upstream, 3′ downstream, and intragenicregions) are made in vitro and then introduced into the desired plantusing standard techniques. Since the efficiency of homologousrecombination is known to be dependent on the vectors used, use ofdicistronic gene targeting vectors as described by Mountford et al.(1994) and Vaulont et al. (1995) may be conveniently used to increasethe efficiency of selecting for altered cap binding protein 80 geneexpression in transgenic plants. The mutated gene interacts with thetarget wild-type gene in such a way that homologous recombination andtargeted replacement of the wild-type gene occurs in transgenic plantcells, resulting in suppression of cap binding protein 80 activity.

Alternatively, oligonucleotides composed of a contiguous stretch of RNAand DNA residues in a duplex conformation with double hairpin caps onthe ends can be used. The RNA/DNA sequence is designed to align with thesequence of the target cap binding protein 80 gene and to contain thedesired nucleotide change. Introduction of the chimeric oligonucleotideon an extrachromosomal T-DNA plasmid results in efficient and specificcap binding protein 80 gene conversion directed by chimeric molecules ina small number of transformed plant cells. This method is described inCole Strauss et al. (1996) and Yoon et al. (1996).

Finally, endogenous genes can also be “knocked-out” by transposons orT-DNA insertion. These are random insertions into the genome thatdisrupt the activity of the endogenous gene into which the transposon orT-DNA is inserted. Since the insertions are random, the subject cells orplants are then screened for those in which cap binding protein 80activity has been reduced.

F. Other Means for Inhibiting can Binding Protein 80 Activity

Cap binding protein 80 activity may be modulated by eliminating theproteins that are required for cap binding protein 80 cell-specific geneexpression. Thus, expression of regulatory proteins, or the sequencesthat control cap binding protein 80 gene expression, or both, can bemodulated using the methods described here.

Another strategy is to inhibit the ability of a cap binding protein 80protein to interact with other proteins. This can be achieved, forinstance, using antibodies specific to cap binding protein 80. In thismethod, cell-specific expression of cap binding protein 80-specificantibodies is used to inactivate functional domains through antibody:antigen recognition (see, Hupp et al., 1995). Interference of activityof a cap binding protein 80 interacting protein can be applied in asimilar fashion.

Alternatively, dominant negative mutants of cap binding protein 80 canbe prepared by expressing a transgene that encodes a truncated capbinding protein 80 protein. Use of dominant negative mutants toinactivate target genes in transgenic plants is described in Mizukami etal. (1996).

In yet another strategy, a peptide, peptidomimetic, or other smallmolecule inhibitor of cap binding protein 80, i.e., an exogenouslyapplied inhibitor, may be identified. Inhibitors may be tested for andscreened by routine assays known in the art. For example, cap bindingprotein 80 may be expressed in plant cells in vitro, and inhibitorsapplied to a culture of cells. Inhibition of cap binding protein 80 maybe analyzed by the “biochemical” and/or “molecular biological” assaysreferred to above. The in vivo analysis of a cap binding protein 80inhibitor may also be conducted, e.g., by applying an inhibitor to aplant and determining if it is effective in inhibiting cap bindingprotein 80 in a plant of interest using assays known to the art. Aneffective inhibition of cap binding protein 80 in a plant cell or plantpreferably results in a decreased level of cap binding protein 80expression, an increased level of ABA, increased anion channel activity,increased drought tolerance, decreased stomatal opening or frequency, orany combination thereof.

Peptide, peptidomimetic, or other cap binding protein 80 inhibitors canbe exogenously applied to the plants. Conveniently, this can be done byadding the inhibitors to irrigation water or to water or to foliarsprays. Inhibitors can be applied alone or in mixture with other planthormones, fertilizers, pesticides or fungicides. The inhibitor can beapplied in a mixture with a carrier or, if necessary, other auxiliaryagents to form any one of the standard types of preparations commonlyused in agriculture, for example, a dry blend, granules, a wettablepowder, an emulsion, an aqueous solution and the like. Suitable solidcarriers are clay, talc, kaolin, bentonite, terra abla, calciumcarbonate, diatomaceous earth, silica, synthetic calcium silicate,kieselguhr, dolomite, powdered magnesia, Fuller's earth, gypsum and thelike. Solid compositions also can be in the form of dispersible powdersor grains, comprising, in addition to the inhibitor, a surfactant tofacilitate the dispersion of the powder or grains in liquid. Liquidcompositions include solutions, dispersions or emulsions containing theauxins together with one or more surface-active agents such as wettingagents, dispersing agents, emulsifying agents, or suspending agents. Inthose applications in which the compounds are applied as a foliar spray,surface-active agents are preferably used. Generally, any number ofsurfactants may be used consistent with the purpose of this constituent.For example the surfactant can comprise a nonionic, anionic, cationic,or zwitterionic surfactant. The surfactant can be formulated with theinhibitor as formulated or, alternatively, the surfactants can beintroduced during application to the plant. In such an instance,regardless of whether the application is conducted via automated ormanual means, the surfactant can be combined with the inhibitor prior toapplication or codispensed separately during application. The averagemolecular weight of useful surfactants ranges from about 100 to about4000. Cationic surfactants useful in compositions of the inventioninclude, for example, amine ethoxylates, amine oxides, mono- anddiilkylamines, imidazolinium derivatives, andalkylbenzyldimethylammonium halides. Nonionic surfactants useful in thecontext of this invention are generally polyether (also known aspolyalkylene oxide, polyoxyalkylene or polyalkylene glycol) compounds.More particularly, the polyether compounds are generallypolyoxypropylene or polyoxyethylene glycol compounds. Anionicsurfactants useful with the invention comprise, for example, alkylcarboxylates, linear alkylbenzene sulfonates, paraffin sulfonates andsecondary n7 alkane sulfonates, sulfosuccinate esters and sulfatedlinear alcohols. Zwitterionic or amphoteric surfactants useful with theinvention comprise a-N-alkylaminopropionic acids,n-alkyl-α-iminodipropionic acids, imidazoline carboxylates, amineoxides, sulfobetaines and sultaines. Although the surfactant can bepresent in the composition in any useful amount, in preferredembodiments, it is present in an amount from about 0.1% to about 25%,more preferably from about 0.1% to about 10% and more preferably stillfrom about 0.5% to about 5%. A surfactant is present in the compositionsof the invention in a useful amount when it facilitates the dissolutionof the inhibitor, or enhances its uptake by the plant, or both, or itseffectiveness in inducing the desired response, or provides all three ofthese effects. Compositions used in the methods of the invention canalso contain suspending agents. Suitable suspending agents are, forexample, hydrophilic colloids, for example polyvinylpyrollidone andsodium carboxymethylcellulose, and vegetable gums, such as gum acaciaand gum tragacanth. Aqueous solutions, dispersions or emulsions may beprepared by dissolving the inhibitor in water or an organic solventwhich can, if desired, contain one or more surface active, sticking,wetting, dispersing, or emulsifying agents. Suitable organic solventsare, for example, alcohols, hydrocarbons, oils and sulfoxides. Inembodiments using alcohols, methanol, isopropyl alcohol, propyleneglycol and diacetone alcohol are preferred. In embodiments using oils,petroleum oils are preferred. Of the sulfoxides, dimethylsulfoxide ispreferred.

The inhibitor can also be microencapsulated. Microcapsules containingthe desired inhibitor can be prepared by co-acervation; or, morepreferably, by stirred interfacial polymerization, e.g., anisocyanate/diamine system. The resulting microcapsules may be used as anaqueous suspension. Inhibitors which are to be used in the form ofaqueous solutions, dispersions or emulsions are generally supplied inthe form of a concentrate containing a high proportion of the inhibitor,and the concentrate is then diluted with water before use. Theseconcentrates are usually required to withstand storage for prolongedperiods and after such storage, to be capable of dilution with water inorder to form aqueous preparations which remain homogeneous for asufficient time to enable them to be applied by conventional sprayequipment. In general, concentrates can conveniently contain from 10–60percent by weight of the inhibitor. Dilute preparations ready for usemay contain varying amounts of the inhibitor. The practitioner canobserve plants treated with the inhibitor and decrease the dilution ofthe inhibitor if it appears the plants are showing signs of wilting orbrowning.

In carrying out the methods of the invention, an “effective amount” of acap binding protein 80 inhibitor is applied to the plants. One of skillwill recognize that an effective amount of an inhibitor will vary andwill depend upon a number of factors including, for example, theparticular inhibitor and formulation selected for use, the timing of theapplication, whether the compound is to be applied for foliar or rootuptake, and the plant species whose growth is to be regulated.

The invention will be further described by the following examples whichare not intended to limit the scope of the invention.

EXAMPLE 1 Isolation of an Oryza sativa Cap Binding Protein 80 (OsCBP80)Gene

It had been reported that a knock out of the Arabidopsis CBP80 generesulted in a plant that had altered hormone signaling, reduced stomatalopening and/or enhanced anion channel activation. Based upon availablehuman CBP80 sequences, Arabidopsis CBP80 sequences in a public databasewere identified. The Arabidopsis CBP80 sequence was then used toidentify contigs in a rice database having CBP80 sequences. Four contigswere identified and oligonucleotide primers (primers 1–15 correspondingto SEQ ID NOs: 3–17) were designed based on the sequences in thosecontigs. Various primer pairs were used in a polymerase chain reaction(PCR) to amplify cDNAs encoding rice CBP80 from a rice root cDNAlibrary. The reaction conditions were 94° C. for 3 minutes; then 35cycles of 94° C. for 0.5 minute and 70° C. for 2.5 minutes; then 4° C.Products were sequenced and a subsequent PCR performed to obtain afull-length clone from the rice root library.

The DNA sequence of the full-length rice CBP80 open reading frame (ORF)and the amino acid sequence of the encoded polypeptide is shown in SEQID NOs:1 and 2, respectively. The ORF begins at position 82 and ends atposition 2688 of the DNA shown in SEQ ID NO:1. As maize, wheat andbanana have sequences with some similarity to SEQ ID NO:1 or 2, thosesequence are useful to isolate orthologs of rice CBP80. Those sequencesinclude SEQ ID Nos: 21–54.

As CBP80 is likely involved in ABA signaling, the rice CBP80 full-lengthDNA or portions thereof are useful to prepare constructs for over orunder expression, or knock outs, or to modify the nucleic acid sequenceso as to encode a CBP80 protein with modified function, i.e., a dominantnegative mutant or a polypeptide lacking or having reduced activity.

Transgenic plants having these constructs have reduced or decreased, orlack, expression of endogenous CBP80, which leads to alterations in ABAsignaling. The alterations in ABA signaling are characterized by reducedstomatal opening and reduced water loss, i.e., the transgenic plantshave enhanced drought tolerance.

EXAMPLE 2 CBP80 Vectors

Overexpression Vectors

Vectors used for expression of full-length genes of interest in plants(overexpression) are designed to overexpress the protein of interest andare of two general types, biolistic and binary, depending on the planttransformation method to be used.

For biolistic transformation, biolistic vectors include i) a backbonewith a bacterial selectable marker (typically, an antibiotic resistancegene) and an origin of replication functional in Escherichia coli (E.coli; e.g., ColE1), and ii) a plant-specific portion including a geneexpression cassette comprising a promoter (e.g., ZmUBlint MOD), the geneof interest (typically, a full-length cDNA) and a transcriptionalterminator (e.g., the Agrobacterium tumefaciens nos terminator), and aplant selectable marker cassette comprising a promoter (e.g., riceActID-BV MOD), a selectable marker gene (e.g., phosphomannose isomerase,PMI) and a transcriptional terminator (e.g., CaMV terminator).

Vectors for transformation by Agrobacterium tumefaciens (A. tumefaciens;binary vectors) include i) a backbone with a bacterial selectable markerfunctional in both E. coli and A. tumefaciens (e.g., spectinomycinresistance mediated by the aadA gene) and two origins of replication,functional in each of aforementioned bacterial hosts, plus the A.lumefaciens virG gene, and ii) a plant-specific portion as describedabove for biolistic vectors except that this portion is flanked by A.tumefaciens right and left border sequences which mediate transfer ofthe DNA flanked by these two sequences to the plant.

Knock Out Vectors

Vectors designed for reducing or abolishing expression of a single geneor of a family of related genes (knock out vectors) are also of twogeneral types corresponding to the methodology used to downregulate geneexpression: antisense or double-stranded RNA interference (dsRNAi).

Antisense Vectors

For antisense vectors, a full-length or partial gene fragment(typically, a portion of the cDNA) can be used in the same vectorsdescribed for full-length expression, as part of the gene expressioncassette. For antisense-mediated downregulation of gene expression, thecoding region of the gene or gene fragment is in the oppositeorientation relative to the promoter; thus, mRNA corresponds to thenon-coding (antisense) strand in planta.

dsRNAi Vectors

For dsRNAi vectors, a partial gene fragment (typically, 300 to 500basepairs long) is used in the gene expression cassette, and isexpressed in both the sense and antisense orientations, separated by aspacer region (typically, a plant intron, e.g., the OsSH1 intron 1, or aselectable marker, e.g., one conferring kanamycin resistance). Vectorsof this type are designed to form a double-stranded mRNA stem, resultingfrom the basepairing of the two complementary gene fragments in plania.

Biolistic or binary vectors designed for overexpression or knock out canvary in a number of different ways, including the selectable markersused in plants and bacteria, the transcriptional terminators used in thegene expression and plant selectable marker cassettes, the methodologiesused for cloning gene or gene fragments of interest (typically,conventional restriction enzyme-mediated or Gateway™ recombinase-basedcloning) and the nature of the gene expression cassette promoter drivingexpression of the gene or gene fragment of interest. For example, thepromoter may drive expression in most tissues of the plants(constitutive, e.g., ZmUBIint MOD), in specific plant tissues (e.g.,maize ADP-gpp for endosperm-specific expression), or in an induciblefashion (e.g., GAL4bsBzl for estradiol-inducible expression in linesconstitutively expressing the cognate transcriptional activator for thispromoter).

For example, a rice CBP80 cDNA or a fragment thereof is subcloned usingconventional restriction enzyme-based cloning into a vector, downstreamof the maize ubiquitin promoter and intron, and upstream of theAgrobacterium tumefaciens nos 3′ end transcriptional terminator. Theresultant gene expression cassette is further subcloned, usingconventional restriction enzyme-based cloning, into the pNOV2117 binaryvector, generating pNOVCBP80.

The pNOVCBP80 binary vector is designed for transformation andoverexpression of CBP80 in monocots. The vector including a binarybackbone containing the sequences necessary for selection and growth inEscherichia coli DH-5α (Invitrogen) and Agrobacterium tumefaciensLBA4404, including the bacterial spectinomycin antibiotic resistanceaadA gene from E. coli transposon Tn7, origins of replication for E.coli (ColE1) and A. tumefaciens (VS1), and the A. tumefaciens vir gene.In addition to the binary backbone, pNOV2117 contains the T-DNA portionflanked by the right and left border sequences, and including thePositech™ (Syngenta) plant selectable marker and the CBP80 geneexpression cassette. The Positech™ plant selectable marker confersresistance to mannose via a cassette including the maize ubiquitinpromoter driving expression of the PMI (phosphomannose isomerase) genefollowed by the CMV transcriptional terminator.

pNOVCBP80 is transformed into a rice cultivar (Kaybonnet) usingAgrobacterium-mediated transformation, and mannose-resistant calli areselected and regenerated. Agrobacterium is grown on YPC solid plates for2–3 days prior to experiment initiation. Agrobacterial colonies aresuspended in liquid MS media to an OD of 0.2 at λ600 nm. Acetosyringoneis added to the agrobacterial suspension to a concentration of 200 μMand the bacteria are induced for 30 minutes.

Three-week-old calli which are induced from the scutellum of matureseeds in the N6 medium (Chu et al., 1975) are incubated in theAgrobacterium solution in a 100×25 petri plate for 30 minutes withoccasional shaking. The solution is then removed with a pipet and thecallus transferred to a MSAs medium which is overlayed with sterilefilter paper. Cocultivation is continued for 2 days in the dark at 22°C. Calli are then placed on MS-Timetin plates for 1 week, after whichthey are transferred to PAA+ mannose selection media for 3 weeks.Growing calli are picked and transferred to PAA+ mannose media andcultivated for 2 weeks in light.

Colonies are transferred to MS20SorbKinTim regeneration media in platesfor 2 weeks in light. Small plantlets are transferred to MS20SorbKinTimregeneration media in GA7 containers. When they reach the lid, they aretransferred to soil in the greenhouse.

Alternatively, plasmid pCIB7613, which contains the hygromycinphosphotransferase (hpt) gene as a selectable marker, may be employedfor transformation. Other useful plasmids include pNADII002(GALA-ER-VP16) which contains the yeast GALA DNA Binding domain (Keeganet al., 1986), the mammalian estrogen receptor ligand binding domain(ER; Greene et al., 1986)) and the transcriptional activation domain ofthe HSV VP16 protein (Triezenberg et al., 1988). Both hpt andGAIA-ER-VP16 are constitutively expressed using the maize Ubiquitinpromoter, and pSGCDL1 (GAL4BS Bz1 Luciferase), which carries the fireflyluciferase reporter gene under control of a minimal maize Bronzel (Bzl)promoter with 10 upstream synthetic GALA binding sites. All constructsuse termination signals from the nopaline synthase gene.

Oryza sativa L. Japonica CV. Taipei 309 is used for the production oftransgenic rice plants. Callus induction, cell suspension, initiation,and maintenance follow protocols previously described by Zhang (1995).Gene transfer is achieved using the Biolistic PDS-1000 system (Bio-Rud.Hercules, Calif.). pCIB7613 is co-transferred with other plasmids in a1:5 molar ratio. DNA coating, high-velocity microprojectile delivery ofDNA, selection, and regeneration of transgenic plants are achievedaccording to Zhang et al. (1998) and Chen et al. (1998a).

Estradiol (Sigma) is resuspended in 95% ethanol and diluted in watercontaining 0.01% Triton X-100 immediately before use. The same volume of95% ethanol is added to the negative control solution without estradiol.Approximately ten-milligram samples of fresh plant tissue are excisedand submerged in estradiol solution, and cultured at 25° C. withoutlight. In whole-plantlet treatments, either intact plantlets aresubmerged in 1 μM estradiol solution and cultured at 25° C. in dark for24 hours, or plantlet roots are submerged in ½ MS salts (Murashige andSkoog, 1962) liquid medium containing estradiol followed by incubationat 25° C. with 16 hour light cycles.

Expression of the CBP80 in transgenic T₀ plants is analyzed. Additionalrice cultivars, including but not limited to, Nipponbare, Taipei 309 andFuzisaka are also transformed and assayed for expression of the CBP80product and enhanced protein expression.

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All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

1. An isolated polynucleotide comprising a nucleotide sequence having anopen reading frame which encodes a rice cap binding protein 80, whichopen reading frame comprises nucleotides 82 to 2688 of SEQ ID NO: 1 orthe complement thereof.
 2. The isolated polynucleotide according toclaim 1, wherein the cap binding protein 80 comprises the amino acidsequence of SEQ ID NO:2.
 3. An expression cassette comprising thepolynucleotide according to claim 1 operably linked to a promoter whichdrives expression in a plant.
 4. The expression cassette according toclaim 3 wherein the open reading frame is in antisense orientationrelative to the promoter.
 5. The expression cassette according to claim3 wherein the open reading frame is in sense orientation relative to thepromoter.
 6. A host cell comprising the expression cassette according toclaim 3, wherein said host cell is a plant cell or bacterial cell.
 7. Atransgenic rice plant, comprising the isolated polynucleotide accordingto claim
 1. 8. A transgenic rice plant, comprising the expressioncassette according to claim
 4. 9. The transgenic rice plant according toclaim 7 which is hypersensitive to ABA compared to a wild-type riceplant.
 10. The transgenic rice plant according to claim 8 which ishypersensitive to ABA compared to a wild-type rice plant.
 11. Arecombinant vector comprising the polynucleotide according to claim 1.12. The recombinant vector according to claim 11 wherein the vector is aplasmid, Ti plasmid, phagemid, cosmid, YAC, BAC, virus, F-factor orphage.
 13. An isolated polynucleotide comprising SEQ ID NO. 1.